Diagnostic assay for Alzheimer&#39;s disease: assessment of Aβ abnormalities

ABSTRACT

The disclosed invention relates to assays for detecting and quantifying Aβ peptide, using solid supports that are coated with heavy metal cations, such as zinc (II) or copper (II) form of a nitriloacetic acid. Further, diagnostic kits are described which are used to carry out the assays of the present invention. An improvement in an assay for detection of Aβ peptide is suggested which comprises forming a heavy metal cation/solid support complex. The preferred heavy metal cations for this improvement are zinc (II) or copper (II) form of a nitriloacetic acid. Finally, methods and kits for bulk purification of Aβ peptides from biological fluids are taught.

This application is a continuation of U.S. Application Ser. No.08/817,423, now U.S. Pat. No. 5,972,634 filed Aug. 4, 1997, which is thenational phase of International Appl. No. PCT/US94/11895, filed Oct. 19,1994.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

Part of the work performed during the development of this inventionutilized U.S. Government Funds under Grants Nos. RO1 NS3048-03 and RO1AG11899-01 from The National Institutes of Health (NIH). The governmentmay have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The purpose of this invention is to assay the quantity and quality of Aβpeptide in Alzheimer's disease (AD) and Aβ amyloidotic disorders relatedto Alzheimer's disease. Specifically, the invention proposes to achievethis end by enriching the peptide by capturing it from biological fluidssuch as plasma, serum, cerebrospinal fluid or urine with a zinc- orcopper-chelated microwell plate, and then measuring the amounts ofcaptured Aβ with specific anti-Aβ antibodies in an ELISA.

2. Related Art

Alzheimer's disease is characterized pathologically by the accumulationin the brain of Aβ protein. The Aβ protein is a small peptide that isalso found cerebrospinal fluid and plasma. Much evidence implicates theaccumulation of Aβ in the pathogenesis of the disease, either as theneurotoxic agent itself or as a hallmark which accompanies neurotoxicityin the disorder. Aβ accumulates as a highly insoluble deposit withinneuronal tissues. It is desirable to discover a treatment which wouldreverse the deposition and relieve or arrest clinical deterioration.

Aggregation of Aβ in the brain is believed to contribute to theprogressive dementia, characteristic of Alzheimer's disease (AD) and tothe premature AD observed among Down's syndrome patients. Aβ, a 4.3-kDapeptide, is the principal constituent of the cerebral amyloid deposits,a pathological hallmark of Alzheimer's disease (AD) (Masters et al.,Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985); Glenner & Wong, Biochem.Biophys. Rev. Commun. 120:885-890 (1984)). Aβ is derived from the muchlarger amyloid protein precursor (APP) (Kang et al., Nature 325:733-736(1987); Tanzi et al., Science 235:880-884 (1987); Robakis et al., Proc.Natl. Acad. Sci. USA 84:4190-4194 (1987); Goldgaber et al., Science235:877-880 (1987)), whose physiological function remains unclear. Thecause of Alzheimer's disease remains elusive; however, the discovery ofmutations of APP close to or within the Aβ domain (Goate et al., Nature349:704-706 (1991); Levy et al., Science 248:1124-1126 (1990); Murrellet al., Science 254:97-99 (1991); Hendricks et al., Nature Genet.1:218-221 (1992), linked to familial AD (FAD) (E. Levy et al., Science248:1124 (1990); Aβ Goate et al., Nature 349:704 (1991); M.Chartier-Harlin et al., Nature 353:844 (1991); J. Murrell, M. Farlow, B.Ghetti, M. D. Benson, Science 254:97 (1991); L. Hendricks et al., NatureGenet. 1:218 (1992); M. Mullan et al., Nature Genet. 1:345 (1992)),indicates that the metabolism of Aβ and APP is likely to be intimatelyinvolved with the pathophysiology of this disorder.

Alzheimer's disease affects 10% of individuals over the age of 60,however, the existence of Aβ deposits in 40% of the brains of normalindividuals in their forties suggests an even larger subclinicalprevalence. Hence, the disease process is likely to be very common, withindividual thresholds of neuronal and functional reserve beingresponsible for the varying onset of clinical symptoms. The disease isdebilitating, chronic, incurable and very expensive to treat and aneffective prevention or therapy would have an enormous commercialmarket. However, there are no reliable biochemical markers for AD.

FAD patients with the “Swedish” APP mutation overproduce the soluble,secreted form of Aβ and suffer from early onset (<60 years) AD (Citronet al., Nature 360:672-674 (1992)). A potential neuropathogenicmechanism has been reported by Younkin and colleagues (Society forNeuroscience, Mol. Genet. Med. 3:95-137 (1993)) for the APP 717mutations which account for 90% of the APP mutations causing FAD. Thesemutations apparently lead to an increase in the ratio of “long” Aβ(1-42) to Aβ (1-40). Aβ (1-40) is the predominant species in thecerebrospinal fluid (CSF), and is a relatively soluble peptide. Aβ(1-42) is significantly more amyloidogenic, and its overproductionrelative to the 1-40 species appears to lead to early-onset AD in thesepatients. Therefore, levels of Aβ (1-40) and Aβ (1-42) in thecerebrospinal fluid (CSF), plasma, serum or urine may be expected tocorrelate with cerebral pathology in sporadic AD cases, the predominantclinical form of the disorder.

Two protocols currently exist for the estimation of Aβ levels inbiological fluids. The first involves the immunoprecipitation of Aβ withspecific anti-Aβ antibodies (e.g. Haass et al., Nature 359:322-325(1992); Citron et al., Nature 360:672-674 (1992)), a technique which is,at best, semiquantitative. This technique was used in combination withwestern blotting to measure Aβ levels in CSF (Shoji et al., Science258:126-129 (1992)) but found no gross differences between AD andcontrol specimens. A double antibody capture ELISA using monoclonalantibodies raised against Aβ appears to give specific Aβ quantificationwith a sensitivity limit at about 0.6 nM (Seubert et al., Nature359:325-327 (1992)).

The double antibody ELISA is more widely used and is the only describedmeans of accurately quantifying Aβ. It has two important limitations. Itrequires an abundance of expensive antibody to coat the wells ofmicrowell plates in order to capture the Aβ from the biological fluid. Asecond anti-Aβ antibody, at a higher dilution, is used to detectcaptured Aβ. The second limitation of the double-antibody capture ELISAtechnique for Aβ assay is that it requires a fluorescence-generatingenzyme-conjugated detection antibody and a fluorescence microwell platereader for the final step of the assay.

Fluorescence plate readers are highly specialized and expensive (about$30,000, Millipore Cytofluor), which limits the accessibility of thetechnique. Fluorescence has been preferred over more versatile, andcheaper, chromogenic assays (e.g., horse radish peroxidase-conjugateddetection antibody acting on a chromogenic substrate), because it lowersthe limit of sensitivity allowing the measurement of Aβ at the levelsfound in biological fluids. No Aβ assay has been described where thedevelopment of a chromogenic substrate was measured by a visible-lightmicrowell plate reader, a far less expensive instrument (e.g., $8,000).

The Aβ species assay of the present invention will provide a rationalbasis to monitor response to putative treatments for AD, as well asproviding early diagnostic information if clinical outcome studiesvalidate the correlation of the Aβ levels in the blood or CSF withdisease progression.

SUMMARY OF THE INVENTION

It has now been found that Aβ strongly and specifically binds zinc andcopper in a pH dependent manner. These binding properties of Aβ havebeen exploited in this invention to create a novel means of capturing Aβfrom biological fluids with a zinc- or copper-treated microwell plate,as well as a novel means for the bulk chromatographic purification of Aβfrom biological fluids.

An advantage of this new ELISA technique over the previously describeddouble antibody capture ELISA is that it obviates the need for a captureantibody (saving reagents and expense) and, because zinc- andcopper-mediated capture appears to be more efficient than immobilizedantibody capture, it is over an order of magnitude more sensitive thanthe reported sensitivity of double antibody capture ELISA. Hence, theassay results with biological fluids can be achieved using cheaperchromogenic substrates, in conjunction with a visible-light microwellplate reader.

In the present invention, an assay is designed to quantify the amount ofAβ peptide present in a solution such as a biological fluid. To do so, asolid substrate is used to which a zinc (II) and/or copper (II) complexis immobilized. Preferably, the metal is complexed with immobilizednitriloacetic acid. The substrate is then contacted with the biologicalfluid. The free coordination sites on the zinc or copper atom act as acapture trap for Aβ peptide which can then be detected and quantified ina number of different ways.

The levels of Aβ are believed to correlate with the cerebral pathologyof AD. However, the more highly amyloidogenic 1-42 species of Aβ may bemore important in AD pathology than other species such as the 1-40 form.The present invention allows levels of the different species of Aβ to bemeasured by use of antibodies that are specific to 1-42 species and donot recognize the 1-40 form. Such antibodies are produced preferrablyfrom mice (although they may be produced from Guinea pigs, rabbits,rats, goats, sheep, horses, et cetera.) by injection with peptidescontaining the unique 40-42 region of the 1-42 species (peptidescomprising the Aβ sequence from residue 42 to any residue less than 38are suitable immunogens). A monoclonal antibody that is specific to the1-42 species of Aβ may be selected by testing for immunoreactivity todifferent Aβ species that have been immobilized on a zinc or coppertreated microwell plate.

Therefore, the first aspect of the invention relates to a diagnosticassay for detecting and/or quantifying Aβ peptide which may be presentin a candidate solution, comprising:

-   -   (a) contacting the candidate solution with a solid support with        a heavy metal cation immobilized thereon to capture Aβ peptide        on the surface of the solid support, thereby forming a first        complex which comprises solid support/heavy metal cation/Aβ        peptide;    -   (b) blocking all exposed metal binding sites remaining after Aβ        capture with a blocker;    -   (c) contacting the first complex, which has been passed through        step (b), with an antibody specific for Aβ peptide to form a        second complex which comprises solid support/heavy metal        cation/Aβ peptide/antibody specific for Aβ peptide;    -   (d) labelling the second complex to form a detectable third        complex which comprises solid support/heavy metal cation/Aβ        peptide/antibody specific for Aβ peptide/label; and    -   (e) detecting the third complex, and quantifying Aβ peptide        which may be present in the candidate solution.

A second aspect of the invention relates to a diagnostic assay fordetecting and/or quantifying Aβ peptide which may be present in acandidate solution, comprising:

-   -   (a) contacting the candidate solution with a solid support with        a heavy metal cation immobilized thereon to capture Aβ peptide        on the surface of the solid support, thereby forming a first        complex which comprises solid support/heavy metal cation/Aβ        peptide;    -   (b) blocking all exposed metal binding sites remaining after Aβ        capture with a blocker;    -   (c) contacting the first complex, which has been passed through        step (b), with an antibody specific for Aβ peptide, called Aβ        antibody, to form a second complex which comprises solid        support/heavy metal cation/Aβ peptide/Aβ antibody;    -   (d) contacting said second complex with one or more        anti-antibodies specific to the Aβ antibody to form a third        complex which comprises solid support/heavy metal cation/Aβ        peptide/Aβ antibody/one or more anti-antibodies;    -   (e) labelling said third complex to form a detectable fourth        complex which comprises solid support/heavy metal cation/Aβ        peptide/Aβ antibody/one or more anti-antibodies/label; and    -   (f) detecting the fourth complex, thereby quantifying Aβ peptide        which may be present in the candidate solution.

The preferred heavy metal cations used in the practice of the presentinvention are zinc (II) or copper (II) complexed to nitriloacetic acid.Other organic ligands which may be used to complex the heavy metal, e.g.copper and zinc, are, but not limited to, iminodiacetic acid,tris(carboxy-methyl)ethylenediamine,N,N,N,N,-carboxy(methyl)tetraethylenepentaminand methionine-polyethyleneglycol (for other such compounds, see F. H.Arnold, Biotechnology 9:151-156 (1991), e.g., at page 154).

The preferred antibodies used in the practice of the invention are thosethat are either specific to Aβ₁₋₄₂ which do not cross react with Aβ₁₋₄₀or specific to Aβ₁₋₄₀ which do not cross react with Aβ₁₋₄₂.

In the preferred embodiments of the invention, the antibodies specificto Aβ protein are labelled with a radioisotope (radioactive isotope),which can then be determined by such means as the use of a gamma counteror a scintillation counter. Isotopes which are particularly useful forthe purpose of the present invention are: ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C,⁵¹Cr, ³⁶Cl, ⁵⁷Co,⁵⁸ Co, ⁵⁹Fe and ⁷⁵Se. In other prefered embodiments ofthe invention, the antibodies specific to Aβ protein are labelled byconjugating them to enzymes which can be detected when conjugated tosaid antibody, such as, but not limited to, fluorescence-generatingenzymes, as well as chromogenic enzymes like alkaline phosphatase,urease, and horseradish peroxidase.

The body fluids that are assayed by the diagnostic assays of the presentinvention, are preferably pretreated as described in the Examples.

Another aspect of the invention relates to kits for carrying out theaforementioned assays which comprise a carrier means, compartmentalizedin close confinement therein to receive one or more container means,which comprises a first container means containing a solid supporthaving a heavy metal cation immobilized thereon and a second containermeans containing an antibody specific for Aβ peptide.

A further aspect of the invention relates to kits for carrying out theabove-mentioned assays, which comprise a carrier means,compartmentalized in close confinement therein to receive one or morecontainer means, which comprises a first container means containing asolid support having a heavy metal cation immobilized thereon, a secondcontainer means containing an antibody specific for Aβ protein, and athird container means containing an anti-antibody which is specific forthe antibody in the second container means. Preferably, theanti-antibody is detectably labeled.

A further aspect of the invention relates to kits preferrably used forcarrying out the above-mentioned assays with biological fluids, whichcomprise a carrier means, compartmentalized in close confinement thereinto receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing an antibodyspecific for Aβ protein, a third container means containing ananti-antibody which is specific for the antibody in the second containermeans, and a fourth container means containing a methylating compound.

Another aspect of the invention relates to kits preferrably used forcarrying out the above-mentioned assays with biological fluids, whichcomprise a carrier means, compartmentalized in close confinement thereinto receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing an antibodyspecific for Aβ protein, a third container means containing ananti-antibody which is specific for the antibody in the second containermeans, a fourth container means containing a methylating compound, and afifth container means containing magnisium chloride.

Another aspect of the invention relates to kits preferrably used forcarrying out the above-mentioned assays with biological fluids, whichcomprise a carrier means, compartmentalized in close confinement thereinto receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing an antibodyspecific for Aβ protein, a third container means containing ananti-antibody which is specific for the antibody in the second containermeans, a fourth container means containing a methylating compound, afifth container means containing magnisium chloride, and a sixthcontainer means containing a blocker.

Yet, another aspect of the invention relates to kits for carrying outthe assays of the present invention which comprises a carrier means,compartmentalized in close confinement therein to receive one or morecontainer means, which comprises a first container means containing asolid support having a heavy metal cation immobilized thereon and asecond container means containing a labelled antibody specific for Aβprotein.

Another aspect of the invention relates to kits preferrably used forcarrying out the assays of the present invention with biological fluidswhich comprises a carrier means, compartmentalized in close confinementtherein to receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing a labelledantibody specific for Aβ protein, and a third container means containinga methylating compound.

A further aspect of the invention relates to kits preferrably used forcarrying out the assays of the present invention with biological fluidswhich comprises a carrier means, compartmentalized in close confinementtherein to receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing a labelledantibody specific for Aβ protein, a third container means containing amethylating compound, and a fourth container means containing magnisiumchloride.

Another aspect of the invention relates to kits preferrably used forcarrying out the assays of the present invention with biological fluidswhich comprises a carrier means, compartmentalized in close confinementtherein to receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing a labelledantibody specific for Aβ protein, a third container means containing amethylating compound, a fourth container means containing magnisiumchloride, and a fifth container means containing a blocker.

The next aspect of the invention relates to kits for carrying out theaforementioned assays which comprise a carrier means, compartmentalizedin close confinement therein to receive one or more container means,which comprises a first container means containing a solid supporthaving a heavy metal cation immobilized thereon and a second containermeans containing an antibody specific for Aβ protein bound to a labelledanti-antibody.

Another aspect of the invention relates to kits preferrably used forcarrying out the aforementioned assays with biological fluids whichcomprise a carrier means, compartmentalized in close confinement thereinto receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing an antibodyspecific for Aβ protein bound to a labelled anti-antibody, and a thirdcontainer means containing a methylating compound.

A further aspect of the invention relates to kits preferrably used forcarrying out the aforementioned assays with biological fluids whichcomprise a carrier means, compartmentalized in close confinement thereinto receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing an antibodyspecific for Aβ protein bound to a labelled anti-antibody, a thirdcontainer means containing a methylating compound, and a fourthcontainer means containing magnisium chloride.

A further aspect of the invention relates to kits preferrably used forcarrying out the aforementioned assays with biological fluids whichcomprise a carrier means, compartmentalized in close confinement thereinto receive one or more container means, which comprises a firstcontainer means containing a solid support having a heavy metal cationimmobilized thereon, a second container means containing an antibodyspecific for Aβ protein bound to a labelled anti-antibody, a thirdcontainer means containing a methylating compound, a fourth containermeans containing magnisium chloride and a fifth container meanscontaining a blocker.

Another aspect of the invention relates to a method for purification ofAβ peptide from biological fluids containing one or more proteins whichcomprises:

-   -   (a) methylating cysteine groups of the proteins in the        biological fluid;    -   (b) acidifying the biological fluid obtained from step (a);    -   (c) applying the biological fluid obtained from step (b) to a        copper-charged chelating-Sepharose column;    -   (d) washing the column with equilibration buffer to obtain an        eluate solution; and    -   (e) collecting the eluate solution, thereby obtaining purified        Aβ peptide.

Another aspect of the invention relates to a method for purification ofAβ peptide from biological fluids containing one or more proteins whichcomprises:

-   -   (a) methylating cysteine groups of the proteins in the        biological fluid;    -   (b) acidifying the biological fluid obtained from step (a);    -   (c) adding to the biological fluid obtained from step (b), a        free copper-charged chelating slurry to form a mixture;    -   (d) centrifuging the mixture obtained from step (c) to obtain a        pellet;    -   (e) washing the pellet obtained from step (d) with equilibration        buffer, thereby obtaining purified Aβ peptide.

A further aspect of the invention relates to a kit for carrying out themethod for bulk purification of Aβ peptide in biological fluids whichcomprises a carrier means compartmentalized in close confinement thereinto receive one or more container means which comprises a first containermeans containing a copper charged chelating-Sepharose column and asecond container means containing an antibody specific for Aβ peptidewhich may be used to confirm presence of purified Aβ peptide.

Finally, another aspect of the invention relates to a kit for carryingout the method of purifying Aβ peptide from biological fluids whichcomprises a carrier means compartmentalized in close confinement thereinto receive one or more container means which comprises a first containermeans containing free copper-charged chelating-Sepharose and a secondcontainer means containing an antibody specific for Aβ peptide which maybe used to confirm presence of purified Aβ peptide.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a, 1 b, 1 c, 1 d and 1 e depict graphs showing analyses of⁶⁵Zn²⁺ binding to Aβ. Values shown are means±S.D., n≧3. (1 a) Scatchardplot. Aliquots of Aβ were incubated (60 min) with ⁶⁵Zn²⁺ in the presenceof varying concentrations of unlabeled Zn²⁺ (0.01-50 μM total). Theproportion of ⁶⁵Zn²⁺ binding to immobilized peptide (1.0 nmol) describedtwo binding curves as shown. The high-affinity binding curve has beencorrected by subtracting the low-affinity component, and thelow-affinity curve has had the high-affinity component subtracted. (1 b)Bar graph showing the specificity of the Zn²⁺ binding site for metals.Aβ was incubated (60 min) with ⁶⁵Zn²⁺ (157 nM, 138,000 cpm) andcompeting unlabeled metal ions (50 μM total). (1 c) Bar graph showing⁶⁵Zn²⁺ (74 nM, 104,000 cpm) binding to negative (aprotinin, insulina-chain, reverse peptide 40-1) and positive (bovine serum albumin (BSA))control proteins and Aβ fragments (identified by their residue numberswithin the Aβ sequence, gln11 refers to Aβ₁₋₂₈ where residue 11 isglutamine). Percent binding of total counts ⁶⁵Zn²⁺/min added iscorrected for the amounts (in nanomoles) of peptides adhering to themembrane. (1 d) Scatchard plot. As for (1 a), with Aβ₁₋₂₈ peptidesubstituting for Aβ₁₋₄₀. 157 nM ⁶⁵Zn (138,000 cpm) is used in thisexperiment to probe immobilized peptide (1.6 nmol). (1 e) Graph showingthe pH dependence of ⁶⁵Zn²⁺ binding to Aβ₁₋₄₀.

FIGS. 2 a, 2 b and 2 c depict graphs showing effect of Zn²⁺ and othermetals on Aβ polymerization using G50 gel filtration chromatography.Results shown are indicative of n≧3 experiments where 55 μg of Aβ isapplied to the column and eluted in 15 ml, monitored by 254 nmabsorbance. (2 a) A graph showing the chromatogram of Aβ in the presenceof EDTA, 50 μM, Zn²⁺, 0.4 μM; Zn²⁺, 25 μM; and Cu²⁺, 25 μM. The elutionpoints of molecular mass standards and relative assignments of Aβ peakelutions are indicated. Mass standards were blue dextran (2×10⁶ daltons,V₀=void volume), BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c(12.4 kDa), and aprotinin (6.5 kDa). The mass of Aβ is 4.3 kDa. (2 b)Bar graph showing the relative amounts (estimated from areas under thecurve) of soluble Aβ eluted as monomer, dimer, or polymer in thepresence of various metal ions (25 μM), varying concentrations of Zn²⁺or Cu²⁺ (the likelihood of Tris chelation is indicated by upper limitestimates), and EDTA. Data for experiments performed in the presence ofcopper were taken from 214 nm readings and corrected for comparison. (2c) Bar graph showing the effects of pre-blocking the chromatographycolumn with BSA upon the recovery of Aβ species in the presence of zinc(25 μM), copper (25 μM), or chelator.

FIGS. 3 a and 3 b depict bar graphs showing Aβ binding to kaolin(aluminum silicate): effects of zinc (25 μM), copper (25 μM), and EDTA(50 μM). (3 a) Bar graph showing the concentration (by 214 nmabsorbance) of Aβ remaining in supernatant after incubation with 10 mgof G50 Sephadex. (3 b) Bar graph showing the concentration (by 214 nmabsorbance) of Aβ remaining in supernatant after incubation with 10 mgof kaolin, expressed as percent of the starting absorbance.

FIGS. 4 a and 4 b depict a blot and a bar graph showing the effect ofZn²⁺ upon Aβ resistance to tryptic digestion. (4 a) A blot of trypticdigests of Aβ (13.9 μg) after incubation with increasing concentrationsof zinc (lane labels, in micromolar), stained by Coomassie Blue.Digestion products of 3.6 kDa (Aβ₆₋₄₀), and 2.1 kDa (Aβ₁₇₋₄₀), as wellas undigested Aβ₁₋₄₀ (4.3 kDa), are indicated on the left. The migrationof the low molecular size markers (STD) are indicated (in kilodaltons)on the right. (4 b) A bar graph showing ⁶⁵Zn²⁺ binding to Aβ trypticdigestion products. The blot was incubated with ⁶⁵Zn²⁺, the visiblebands excised, and the bound counts for each band determined. These dataare typical of n=3 replicated experiments.

FIG. 5 depicts a graph showing a scatchard analysis of ⁶⁵Zn binding torat Aβ₁₋₄₀. Dissolved peptides (1.2 nMol) were dot-blotted onto 0.20μPVDF membrane (Pierce) and competition analysis performed as describedin Example 1 (FIG. 1). Rat Aβ₁₋₄₀ and human Aβ₁₋₄₀ were synthesized bysolid-phase Fmoc chemistry. Purification by reverse-phase HPLC and aminoacid sequencing confirmed the synthesis. The regression line indicates aK_(A) of 3.8 μM. Stoichiometry of binding is 1:1. Although the datapoints for the Scatchard curve are slightly suggestive of a biphasiccurve, a biphasic iteration yields association constants of 2 and 9 μM,which does not justify an interpretation of physiologically separatebinding sites.

FIGS. 6 a, 6 b, 6 c and 6 d depict graphs showing the effect of zincupon human, ¹²⁵I-human and rat Aβ₁₋₄₀ aggregation into >0.2μ particles.Stock human and rat Aβ₁₋₄₀ peptide solutions (16 μM) in water werepre-filtered (Spin-X, Costar, 0.2μ cellulose acetate, 700 g), brought to100 mM NaCl, 20 mM Tris-HCl, pH 7.4 (buffer 1)±EDTA (50 μM) or metalchloride salts, incubated (30 minutes, 37° C.) and then filtered again(700 g, 4 minutes). The fraction of the Aβ₁₋₄₀ in the filtrate wascalculated by the ratio of the filtrate OD₂₁₄ (the response of theOD₂₁₄, titrated against human and rat Aβ₁₋₄₀ concentrations (up to 20 μMin the buffers used in these experiments), was determined to be linear)relative to the OD₂₁₄ of the unfiltered sample. All data points are intriplicate, unless indicated. (6 a) A graph showing the proportions ofAβ₁₋₄₀, incubated±Zn²⁺ (25 μM) or EDTA (50 μM) and then filtered through0.2μ, titrated against peptide concentration. (6 b) A graph showing theproportion of Aβ₁₋₄₀ (1.6 μM) filtered through 0.2μ, titrated againstZn²⁺ concentration. ¹²⁵I-human Aβ₁₋₄₀ (²⁵I-human Aβ₁₋₄₀ was preparedaccording to the method in Mantyh et al., J. Neurochem 61:1171 (1993)(15,000 CPM, the kind gift of Dr. John Maggio, Harvard Medical School)was added to unlabeled Aβ₁₋₄₀ (1.6 μM) as a tracer, incubated andfiltered as described above. The CPM in the filtrate and retained on theexcised filter were measured by a γ-counter. (6 c) A bar graph showingthe proportion of Aβ₁₋₄₀ (1.6 μM) filtered through 0.2μ followingincubation with various metal ions (3 μM). The atomic number of themetal species is indicated. (6 d) A graph showing the effects of Zn²⁺(25 μM) or EDTA (50 μM) upon kinetics of human Aβ₁₋₄₀ aggregationmeasured by 0.2μ filtration. Data points are in duplicate.

FIGS. 7 a, 7 b, 7 c and 7 d depict bar graphs showing the sizeestimation of zinc-induced Aβ aggregates. (7 a and 7 b) Bar graphsshowing the proportion of Aβ₁₋₄₀ (1.6 μM in 100 mM NaCl, 20 mM Tris-HCl,pH 7.4 (buffer 1), incubated±Zn²⁺ (25 μM or EDTA (50 μM) and thenfiltered through filters of indicated pore sizes (Durapore filters(Ultrafree-MC, Millipore) were used for this study, hence there is aslight discrepancy between the values obtained with the 0.22μ filters inthis study compared to values obtained in FIG. 6 using 0.2μ Costarfilters). (7 c) A bar graph showing ⁶⁵ZnCl₂ (130,000 CPM, 74 nM) used asa tracer of the assembly of the zinc-induced aggregates of human Aβ₁₋₄₀produced in FIGS. 7 a and 7 b. By determining the amounts of Aβ₁₋₄₀ and⁶⁵Zn in the filtrate, the quantities retarded by the filters could bedetermined, and the stoichiometry of the zinc: Aβ assemblies estimated.(7 d) Bar graph. Following this procedure, the filters, retaining Zn: Aβassemblies, were washed with buffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH7.4)+EDTA (50 μM×300 μl, 700 g, 4 minutes). The amounts ofzinc-precipitated Aβ₁₋₄₀ resolubilized in the filtrate fraction weredetermined by OD₂₁₄, and expressed as a percentage of the amountoriginally retained by the respective filters. ⁶⁵Zn released into thefiltrate was measured by γ-counting.

FIGS. 8 a and 8 b are photographs showing zinc-induced tinctorialamyloid formation. (8 a) Zinc-induced human Aβ₁₋₄₀ precipitate stainedwith Congo Red. The particle diameter is 40μ. Aβ₁₋₄₀ (200 μl×25 μM inbuffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH 7.4)) was incubated (30minutes, 37° C.) in the presence of 25 μM Zn²⁺. The mixture was thencentrifuged (16,000 g×15 minutes), the pellet washed in buffer 1 (100 mMNaCl, 20 mM Tris-HCl, pH 7.4)+EDTA (50 μM), pelleted again andresuspended in Congo Red (1% in 50% ethanol, 5 minutes). Unbound dye wasremoved, the pellet washed with buffer 1 (100 mM NaCl, 20 mM Tris-HCl,pH 7.4) and mounted for microscopy. (8 b) The same aggregate visualizedunder polarized light, manifesting green birefringence. The experimentwas repeated with EDTA (50 μM) substituted for Zn²⁺ and yielded novisible material.

FIG. 9 depicts a graph showing the effect of zinc and copper upon human,¹²⁵I-human and rat Aβ₁₋₄₀ aggregation into >0.2μ particles. Stock humanand rat Aβ₁₋₄₀ peptide solutions (16 μM) in water were pre-filtered(Spin-X, Costar, 0.2μ cellulose acetate, 700 g), brought to 100 mM NaCl,20 mM Tris-HCl, pH 7.4 (buffer 1)±EDTA (50 μM) or metal chloride salts,incubated (30 minutes, 37° C.) and then filtered again (700 g, 4minutes). The fraction of the Aβ₁₋₄₀ in the filtrate was calculated bythe ratio of the filtrate OD₂₁₄ (the response of the OD₂₁₄, titratedagainst human and rat Aβ₁₋₄₀ concentrations (up to 20 μM in the buffersused in these experiments), was determined to be linear) relative to theOD₂₁₄ of the unfiltered sample. All data points are in triplicate,unless indicated. (FIG. 9) The graph shows the proportions of Aβ₁₋₄₀,incubated±Zn²⁺ (25 μM) or Cu²⁺ or EDTA (50 μM) and then filtered through0.2μ, titrated against peptide concentration.

FIG. 10 depicts the amino acid sequence of human Aβ peptide. The aminoacid sequence of human Aβ peptide (SEQ ID NO:1) is depicted and aminoacid positions are numbered.

FIG. 11 depicts a standard curve graph for increasing Aβ concentrationon an ELISA using a copper coated 96-well plate for solid phase capture.Values are shown±S.D. n=3. Increasing concentrations of Aβ were preparedin coating buffer (Tris 20 mM, pH 7.4 and NaCl 150 mM). Aliquots (200μl) were transferred to the wells of a copper-treated 96-well plate((copper (II) was immobilized on the well surface with nitriloaceticacid) and incubated for 2 h at 37° C. The solution in the wells wasremoved and replaced with 300 μl per well of blocking buffer (2% gelatinin Tris 20 mM, pH 8 and NaCl 150 mM) and the plate incubated at 37° C.for a further 2 h. The wells were washed two times with 300 μl aliquot'sof washing buffer (Tris 20 mM, pH 8 and NaCl 150 mM) before beingincubated (2 h at 37° C.) with a primary antibody (200 μl per well ofantibody diluted 1/1000 with blocking buffer containing a reducedgelatin concentration (0.2%)) directed at the N-terminus of Aβ (a nowcommercially available mouse monoclonal antibody supplied by Dr. S. K.Kim of the NY State Institute for Basic Research in DevelopmentalDisabilities). The wells were washed three times with washing bufferbefore the addition of anti-mouse-antibody-HRPO conjugate (200 μl perwell of a 1/1000 dilution in 0.2% gelatin blocking buffer) and a finalincubation at 37° C. for 2 h. The wells were washed three times withwashing buffer and one final rinse in water before the addition of 200μl per well of HRPO substrate solution (Pierce, 34024). After a 30minute incubation at room temperature (18-22° C.) a 25 μl aliquot of 2 MH₂SO₄ was added to each well and absorbance of the plate measured at 450nm. The average background absorbance (wells containing no Aβ) wassubtracted from the absorbance of Aβ standards and the resulting valuesplotted against peptide concentration.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Aβ₁₋₄₀, the major component of Alzheimer's disease cerebral amyloid isrelatively soluble at high concentrations (≦3.7 mM) and has beendetected in CSF and blood. Physiological factors which decreasesolubility and induce Aβ amyloid formation may be important in thepathogenesis of the disease. It has been discovered that human Aβspecifically and saturably binds zinc, and that concentrations of thismetal ion above 300 nM rapidly destabilize human Aβ₁₋₄₀ solutions,inducing tinctorial amyloid formation. High-affinity binding (K_(A)=107nM) compatible with normal CSF zinc levels, and low-affinity binding(K_(A)=5.2 μM) have now been shown. In contrast, rat Aβ₁₋₄₀ binds zincless avidly and does not aggregate in its presence, suggesting apossible explanation for lack of cerebral Aβ amyloid in these animals.Collectively, these data suggest a potentially critical role forcerebral zinc metabolism in the neuropathogenesis of Alzheimer'sdisease.

Further, it has been observed that abnormalities of zinc homeostasisoccur in AD and DS patients. Cerebral zinc homeostasis, which has beenreported to be abnormal in AD (D. Wenstrup, W. D. Ehmann, W. R.Markesbery, Brain Res. 533:125 (1990); J. Constantinidis, Encephale16:231 (1990); F. M. Corrigan, G. P. Reynolds, N. I. Ward, Biometals6:149 (1993); C. O. Hershey et al., Neurology 33:1350 (1983)) may beimportant for the metabolic fate of Aβ since increased concentrations ofzinc promote the peptide's adhesiveness and resistance to proteolyticdigestion. Moreover, oral zinc supplementation has recently been shownto have an acutely adverse effect on cognition in AD subjects, but notage-matched controls indicated that environmental or nutritional zincexposure may be a contributing factor to AD pathophysiology.

The present findings have indicated that Aβ strongly and specificallybinds zinc in a pH dependent manner. In the brain milieu, these metalions are present in sufficient concentration to exert these effects onbinding and solubility. A decrease in Aβ solubility occurs in thepresence of concentrations of zinc as low as 0.3 μM. Occupation of thezinc binding site on Aβ increases the resistance of the peptide totryptic digestion at the α-secretase site. α-Secretase is an, as yet,unidentified protease which has been observed to cleave the precursormolecule of Aβ, the Amyloid Protein Precursor (APP) within the Aβdomain, rendering Aβ incapable of accumulating. Hence, occupation of thezinc binding site on Aβ will increase the biological half-life of thepeptide and so increase its availability for deposition.

The diagnostic assays of the present invention are carried out asexemplified in Example 14, below. In general, a commonly used protocolfor an ELISA is followed. The Aβ peptide acts as the antigen of aconventional direct ELISA. The plates used are coated with zinc (II) orcopper (II), hence, enabling the relatively stable binding of the Aβpeptide to the surface of the plate. Preferably, the zinc (II) or copper(II) is complexed to a ligand which is immobilized on the plate.Examples of such ligands include nitriloacetic acid and iminodiaceticacid. The complexes are prepared by dissolving the ligand in an organicsolvent such as ether, depositing the solution on a solid support,letting the solvent evaporate, and then adding an equeous solution of azinc (II) or copper (II) salt (such as the chloride). The solid supportmay then be washed with additional solution to remove unreacted ligandand metal salt. Preferred solid supports include but are not limited tonitrocellulose, diazocellulose, microtiter plates, glass, plastic,polystyrene, polyvinyl, polyvinylchloride, polypropylene, polyethylene,dextran, affinity support gels such as Sepharose or agar, starch, andnylon. Those skilled in the art will note that many other suitablecarriers for binding the ligand exist, or will be able to ascertain thesame by use of routine experimentation.

In a different embodiment of the diagnostic assays, the Aβ peptide in asolution can be detected by using solid support particles. Theparticles, beads or pieces of a solid support, are coated with zinc (II)or copper (II) form of nitriloacetic acid, thus, enabling the relativelystable binding of the Aβ peptide to the surface of the particle. Thecandidate solution is added to the particles and incubated asbefore-described to allow binding of the Aβ peptides to the surface(s)of the particles. Labelled antibodies, particularly radiolabelled ones,are used to bind to the Aβ peptides. Alternatively, antibodies specificfor Aβ are added to bind to the Aβ peptides, and then labelledanti-antibodies, particularly radiolabelled ones, which are specific forthe Aβ antibodies, are added to bind to the Aβ antibodies, therebyallowing detection and quantification of the Aβ peptides. The Aβpeptides are detected and/or quantified with the appropriate means,e.g., scintillation counter.

The bulk purification of Aβ from biological fluids is best achieved withcopper charged chelating-Sepharose (Pharmacia, catalog no. 17-0575-01).The cysteine groups in the sample proteins are first methylated withN-methyl maleimide, about 1-20 mM, preferrably, about 10-20 mM, and mostpreferrably, about 10 mM for about 1-2 hours, preferrably about 1 hour,(other appropriate compounds, such as, iminodiacetic acid, may be usedinstead of maleimide in simillar concentrations and for simillar periodsof time), then acidified by titrating pH to about 4.9-5.0, preferrablyto about 5.0, using about 1-2 M, preferrably about 1M, sodium acetate,pH about 3-4, preferrably about 3.5, and the total NaCl concentrationincreased by about 450-550 mM, preferrably by about 500 mM, with about4-5 M, preferrably about 5M NaCl. The sample is then applied to acopper-charged chelating-Sepharose column (e.g., 250 μl bed volume forabout 15 ml of CSF) or free copper-charged chelating-Sepharose slurry(about 50-60 μl, preferrably about 50 μl of about 50% v/v) is added tothe sample if the volume is less than about 4 ml. Equilibration bufferis about 450-550 mM, preferrably about 500 mM NaCl about 25-100 mM,preferrably about 50 mM MES, pH about 4.0-5.1, preferrably about 5.0 andis used to wash the column or the Sepharose pellet followingcentrifugation (preferably, low speed centrifugation (about 1,200-1,800g, preferrably about 1,500 g, for about 2-4 minutes, preferrably about 3minutes)). It should be noted that as the speed of centrifugationincreases, the centrifugation time decreases. The Sepharose pellet isdeveloped with SDS sample buffer containing 50 mM EDTA if the sample isto be applied in entirety to western blot analysis. Alternatively, theSepharose can be developed with about 450-550 mM, preferrably about 500mM NaCl, 50 mM EDTA, pH about 7.0-9.0, preferrably about 8.0, alone andthe eluate sampled for western blot analysis. The treatment of 15 ml ofCSF by this method enriched both soluble APP as well as 4.3 and 3.6 kDaspecies of Aβ (identified by an antibody that identifies an epitope inthe first 16 residues of Aβ; commercially available). In order to bindcopper or zinc, the peptide requires an intact domain from residues6-28. 4G8 only recognized the two Aβ species and not APP, confirmingthat the APP captured by the Sepharose was post-secretase cleavedsoluble APP. The use of specific anti-Aβ antibodies as described aboveon western blot analysis of these products can confirm the specificityof the ELISA immunoreactivity.

Definitions

Aβ peptide is also known in the art as Aβ, β protein, β-A4 and A4.

Amyloid as is commonly known in the art, and as is intended in thepresent specification, is a form of aggregated protein.

Similarly, Aβ Amyloid is an aggregated Aβ peptide. It is found in thebrains of patients afflicted with AD and DS and may accumulate followinghead injuries and in Guamanian amyotrophic lateral sclerosis/Parkinson'sdementia (GALS/PDC).

Tinctorial amyloid is referred to amyloid that in addition to beinginsoluble in aqueous buffer can be stained with Congo Red, and haspositive birefringence in polarized light.

Anti-amyloidotic agent refers to a compound that inhibits formation ofamyloid.

Zinc-induced Aβ aggregates are, like tinctorial amyloid, insoluble inaqueous buffer and stain with Congo Red.

Aβ amyloidosis, as is commonly known in the art and intended in thepresent specification, refers to the pathogenic condition in humans andother animals which is characterized by formation of Aβ amyloid inneural tissue such as brain.

Pre-filtering and pre-filtered as used in the present specificationmeans passing a solution, e.g. Aβ peptide in aqueous solution, through aporous membrane by any method, e.g. centrifugation, drip-through bygravitational force, or by application of any form of pressure, such asgaseous pressure.

Physiological solution as used in the present specification means asaline solution which comprises compounds at physiological pH, about7.4, which closely represents a bodily or biological fluid, such as CSF,blood, plasma, et cetera.

Heavy metal chealating agent refers to any agent, e.g., compound ormolecule, which chelates heavy metals, i.e., binds the heavy metal verytightly and can inhibit or stop interaction with other agents. Examplesof such heavy metal chealating agents are EDTA or Desferrioxamine.

In the present invention, the heavy metal salts are of any heavy metalor any transition metal, in any form, soluble or insoluble, e.g. thechloride, bromide, or iodide salts.

A blocker of heavy metal cations as used in the present invention refersto any compound that binds to all exposed metal binding sites remainingon the heavy metal cations, which are conjugated to the solid support,after Aβ capture. Examples of such blockers are, but are not limited to,gelatin (Biorad, catalog no. 170-6537) and SuperBlock (Pierce, catalogno. 375-35).

In the present specification, unless otherwise indicated, zinc meanssalts of zinc, i.e., Zn²⁺ in any form, soluble or insoluble.

Biological fluid means fluid obtained from a person or animal which isproduced by said person or animal. Examples of biological fluids includebut are not limited to cerebrospinal fluid (CSF), blood, serum, urine,and plasma. In the present invention, biological fluid includes whole orany fraction of such fluids derived by purification by any means, e.g.,by ultrafiltration or chromatography.

Neat sample of a biological fluid means that the biological fluid hasnot been altered by, for example, dilution.

Control human subject refers to a healthy person who is not afflictedwith amyloidosis.

Synthetic peptide standard in the present invention means an assembly ofamino acids linked by peptide bonds that is synthesized in a laboratory.Methods for making synthetic peptides include, although are notrestricted to, such procedures as solid phase P_(moc) chemistry.

A candidate solution in the present invention means a solution which issuspected of containing Aβ peptide.

An anti-antibody in the present invention means an antibody that bindsspecifically to another antibody. Generally such antibodies are obtainedby immunizing an animal with the antibody from another animal. Thus, onecan obtain goat anti-IgG polyclonal antibodies in this way.

A solid support in the present invention means any solid material towhich the heavy metal cations can be complexed, and which can be used tomake and use the invention. Examples of such solid support are, but notlimited to, microtitre plates, petri dishes, bottles, slides, and othersuch containers made of plastic, glass, polyvinyl, polystyrene, andother solid materials which do not interfere with the formation ofcomplexes and allow detection of labelled antibodies.

More specifically, solid support particles which may be used in thepresent invention are irregular shaped solid supports such as beads,particles, and pieces of the aforementioned solid materials which may beused for the practice of the present invention.

Persons skilled in the art are able to screen for and determine theusefulness of a solid support material by parallel testing andcomparison between the material in question and a known solid supportmaterial such as polyvinyl or polystyrene.

In the present invention, the Aβ peptide may be comprised of anysequence of the Aβ peptide as long as it contains at least the aminoacids corresponding to positions 6 through 28 of Aβ peptide whichcomprise the binding site for zinc, the most preferred heavy metalcation capable of binding to a polypeptide comprising at least aminoacids 6 to 28 of Aβ. The preferred embodiments of the invention make useof peptides Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁, Aβ₁₋₄₂, and Aβ₁₋₄₃. The mostpreferred embodiment of the invention makes use of Aβ₁₋₄₀. However, anyof the Aβ peptides which comprises at least amino acids 6 to 28 of Aβmay be employed according to the present invention. The sequence of Aβpeptide, including amino acids 6 to 28, is found in FIG. 10 (C. Hillbichet al., J. Mol. Biol. 228:460-473 (1992)).

In the present method, the Aβ peptide is directly detected by usingoptical spectrophotometry. This is possible because a direct correlationexists between concentration of the peptide and OD₂₁₄ measurements.Although the preferred wave length for the OD measurements is about 214nm, the measurements may be carried out for the purpose of the presentinvention at wave lengths from about 190 to about 500 nm. Preferred wavelengths are, however, from about 208 to about 280 nm.

Further, the Aβ peptide may be detected by radiolabelling the peptideand measuring the compounds per minute (CPM) of the filtrates and/or thepellets. A preferred radiolabelled Aβ peptide in the present inventionis ³H-Aβ. Other radiolabels which can be used in the present inventionare ¹⁴C and ³⁵S.

The labelled antibodies and anti-antibodies are detected by usingvisible-light microwell plate reader (for chromogenic enzymes),fluorescence microwell plate reader (for fluorescence-generatingenzymes), and scintillation counter (for radioisotopes). The types oflabels and the appropriate means for detectioin of the labels, however,are not limited to those specifically mentioned herein.

Other heavy metal cations capable of binding to a polypeptide comprisingat least amino acids 6 to 28 of Aβ which may be used in the practice ofthe invention include metallochloride salts, preferably of zinc, copper,or mercury. The most preferred embodiment of the invention, however,makes use of zinc chloride.

In the preferred embodiments of the invention, the antibodies specificto Aβ protein are labelled with a radioisotope (radioactive isotope),which can then be determined by such means as the use of a gamma counteror a scintillation counter. Isotopes which are particularly useful forthe purpose of the present invention are: ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵ S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁹Fe and ⁷⁵Se.

Another way in which the antibody of the present invention can bedetectably labeled is by linking or conjugating the same to an enzyme.This enzyme, in turn, when later exposed to its substrate, will reactwith the substrate in such a manner as to produce a chemical moietywhich can be detected as, for example, by spectrophotometric,fluorometric or visual means. Examples of enzymes which can be used todetectably label the antibody of the present invention include malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,catalase, glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholine esterase. Avidin-biotin binding may be used to facilitatethe enzyme labeling.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due to thefluorescence of the dye. Among the most commonly used fluorescentlabelling compounds are fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

The antibody of the invention can also be detectably labeled usingfluorescent emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the antibody molecule using suchmetal chelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The antibody of the present invention also can be detectably labeled bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged antibody is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent antibody is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Another technique which may also result in greater sensitivity when usedin conjunction with the present invention consists of coupling theantibody of the present invention to low molecular weight haptens. Thehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin (reacting withavidin) or dinitrophenyl, pyridoxal and fluorescamine (reacting withspecific anti-hapten antibodies) in this manner.

In addition, the sensitivity of the assay may be increased by use ofamplification strategies including substrate cycling and enzymechanneling as taught by Mosbach (Lindbladh et al., Trends in Biochem.Sci. 18:279-283 (1993).

The pH of the reaction mixtures for Aβ capture to immobilized metal ionsis, unless otherwise indicated, preferably close to neutral (about 7.4).The pH, therefore, may range from about 6.8 to about 8.5, preferablyfrom about 7 to about 7.8, and most preferably about 7.4. The pH ofother incubations, including antibody and anti-antibody incubations isbetween 7-9, preferrably at 8.

Buffers which can be used in the methods of the present inventioninclude, but are not limited to, Tris-chloride and Tris-base, MOPS,HEPES, bicarbonate, Krebs, and Tyrode's. The concentration of thebuffers may be between about 10 mM and about 500 mM. However,considering that these buffers chelate zinc, the concentration of thebuffers should be kept as low as possible without compromising theresults.

The present invention permits use of very low concentrations of Aβpeptide, e.g. from about 0.1 nM to 3.7 mM (upper limit of solubility). Apreferred embodiment of the invention employs about 0.8 nM concentrationof Aβ peptide, the lowest detectable concentration of Aβ previouslyreported for an ELISA type assay was 0.5 nM (Schubert et al., Nature359:325-327 (1992)).

The present invention may be practiced at temperatures ranging fromabout 1 degree centigrade to about 99 degrees centigrade. The preferredtemperature range is from about 4 degrees centigrade to about 40 degreescentigrade. The most preferred temperature for the practice of thepresent invention is about 37 degrees centigrade. Therefore, anadvantage of the present invention is the greatest sensitivity overprevious detection systems.

The Aβ peptide is trapped by the free coordination sites on the zinc orcopper atoms (binds to the zinc or copper atoms) at near-instantaneousrate. However, defusion rates are a limiting factor in the absorption ofthe peptide and antibodies to the solid phase. In a preferred embodimentof the invention, the incubations are carried out for about 90-240,preferably about 120, minutes to maximize capture.

To determine whether Aβ binds zinc, a synthetic peptide representingsecreted Aβ₁₋₄₀ was incubated with ⁶⁵Zn²⁺. Rapid binding (60% B_(max) at1 min), which plateaued at 1 h, was observed. Scatchard analysis of⁶⁵Zn²⁺ binding describes two saturable binding curves, a high affinitycurve (K_(a)<107 nM), and a lower affinity curve (K_(a)<5.2 μM) FIG. 1a). The affinity constant estimates might be skewed by assuming that theTris buffer does not bind zinc. In fact, Tris-HCl binds zinc and copperwith stability constants of 4.0 and 2.6, respectively (Dawson et al.,Data for Biochemical Research, Oxford University Press (1986)).Incubating Aβ in the presence of higher concentrations of Tris (150 and500 mM) abolishes ⁶⁵Zn²⁺ binding to Aβ (≈50% and ≈95%, respectively),indicating that Tris-induced Zn²⁺ chelation cannot be excluded. Ourcalculated affinity constants are therefore upper limit estimates.

⁶⁵Zn²⁺ binding is very specific, with Zn²⁺ being the only unlabeledmetal ion tested that is capable of competing off the label (FIG. 1 b).To determine the specific region of Aβ involved in zinc binding and tovalidate the dot-blot binding system, equivalent amounts of variouspeptides representing fragments of Aβ₁₋₄₀ and peptide controls wereassayed for ⁶⁵Zn²⁺ binding in this system (FIGS. 1 c and 1 d).

The reverse sequence (40-1) control peptide only binds 50% of B_(max)compared with Aβ₁₋₄₀ (FIG. 1 c), indicating that zinc binding is notmerely a consequence of the presence of favorable residues. Aβ₁₋₂₈ bound30% of B_(max), indicating that the carboxyl terminus plays an importantrole in promoting zinc binding. Glutamine substitution for the glutamateat position 11 of Aβ₁₋₂₈, in accordance with the Down's syndrome Aβsequence reported by Glenner and Wong, Biochem. Biophys. Res. Commun.120:885-890 (1984), does not interfere with ⁶⁵Zn²⁺ binding. TheScatchard plot of ⁶⁵Zn²⁺ binding to Aβ₁₋₂₈ reveals similar low-affinity(K_(a)<15 μM) and high-affinity (K_(a)<334 nM) binding associations(FIG. 1 d) to those of Aβ₁₋₄₀, but overall the Aβ₁₋₂₈ peptide binds zincless avidly. Although the Aβ₁₋₂₈ peptide clearly binds zinc, peptidesoverlapping this region (1-17 and 12-28) do not individually bind zinc.Additionally, a peptide covering a region of the carboxyl terminus(25-35) also is unable to bind zinc (FIG. 1 c).

The calculated stoichiometry of high-affinity Zn²⁺-binding to Aβ,derived from the x-intercepts on the Scatchard plots (FIG. 1, a and d),is 0.7:1 (Aβ₁₋₄₀) and 1:4 (Aβ₁₋₂₈). For low-affinity binding, theZn²⁺:Aβ ratio is 2.5:1 (Aβ₁₋₄₀) and 4:1 (Aβ₁₋₂₈).

⁶⁵Zn²⁺ binding of sequenced tryptic digest products of Aβ (FIG. 4 b)indicates that the 6-40 fragment binds zinc, but that the other visibledigest fragment 17-40 (FIG. 4 b), equivalent to the post-secretase (Eschet al., Science 248:1122-1124 (1990); Sisodia et al., Science248:492-495 (1990)) carboxyl-terminal product produced in vivo, does notbind zinc. The contribution of histidines (residues 6, 13, and 14) toZn²⁺ binding is indicated by the deterioration of binding with lower pH(30% of B_(max) at pH 6.0, FIG. 1 e). Taken together, these dataindicate that zinc coordination requires the contiguous sequence betweenresidues 6 and 28, a region containing all 3 histidine residues, andthat optimal zinc binding also requires the presence of thecarboxyl-terminal domain.

Further experiment investigated whether zinc binding could affect Aβconformation as assayed by migration on gel-filtration chromatography.Major Aβ species believed to correspond to monomeric, dimeric, andpolymeric forms were observed (FIG. 2 a). Total concentrations of Zn²⁺as low as 0.4 μM decrease recovery of Aβ compared with elution profilesobtained in the presence of EDTA and other metals (FIGS. 2 a and 2 b).At 25 μM total Zn²⁺, <20% of the Aβ applied to tthe column is eluted.The greatest loss occurred among high order polymer and dimeric species.The relative amount of monomeric Aβ is less affected. A systematicassessment of several metals indicates that the reduction of Aβrecoverable by chromatography is most sensitive to Zn²⁺, with relatedtransition metals Co²⁺, Ni²⁺, and Fe²⁺ (at 25 μM) displaying similareffects on chromatography to those obtained with only 10 μM Zn²⁺ (FIG. 2b). Other transition metals, heavy metals, and Al³⁺ (25 μM) have partialeffects on Aβ solubility comparable with 3 μM total Zn²⁺. Meanwhile,Ba²⁺, Ag²⁺, Mg²⁺, and Ca²⁺ (25 μM) have the least effect on Aβ comparedwith the EDTA profile, although a 60% reduction in eluted peptide wasobserved in the presence of these metal ions. Pb²⁺ (25 μM) most stronglypromotes the elution of the monomeric peptide, abolishing high orderpolymers; overall recovery is similar to that obtained with 0.4 μM totalZn²⁺. In making comparisons of the effects of these metal ions, it isagain important to consider the differential metal ion chelating effectsof Tris previously mentioned.

A dramatic increase in Aβ dimerization is observed with Cu²⁺ (25 μMtotal). This metal also induces exaggerated Aβ absorbance (4-fold) at254 nm when compared with 214 nm absorbance and induces the monomericspecies to apparently fluoresce at 254 nm causing negative readings(FIG. 2 a) which are proportionally positive at 214 nm (FIG. 2 b). Ahigher concentration of Cu²⁺ (80 μM total) promotes increased recoveryof Aβ, indicating that the presence of Cu²⁺ at relatively lowconcentrations (less than 25 μM) favors solubility in this system.

The metal ions which most favored Aβ solubility (Mg²⁺, 25 μM and totalCu²⁺, 25 μM) were tested for their ability to stabilize Aβ in a solublestate in the presence of 25 μM total Zn²⁺. These combinations neitherrescue nor worsen Zn²⁺-induced loss of Aβ recovery (FIG. 2 b). Overall,these data suggest that Zn²⁺ reduces the recovery of Aβ, whereas achelating agent attenuates this effect.

Chromatography of Aβ was performed under various conditions to determineif zinc-induced loss of Aβ could be blocked. Pretreating the column with3% BSA as an adsorption blocker significantly increased the amounts ofAβ recovered from the column, and suggest that on untreated apparatusthe peptide precipitates onto a column component (FIG. 2 c). Blockingthe column results in a 200% increase in the recovery of Aβ in thepresence of Zn²⁺ (25 μM total), a 75% increase in recovery in thepresence of Cu²⁺ (25 μM total), but only a 10% increase in the presenceof EDTA (50 μM). This indicates that precipitation onto the column ismost specifically accelerated by zinc.

To determine the part of the column onto which Aβ was precipitating, Aβsolutions were incubated with various column components and assayed byUV absorption before and after incubation. Replicating thechromatography experimental conditions, Aβ (100 μM in equilibrationbuffer) was incubated for 1 h in plastic reaction vessels with orwithout the presence of Sephadex. Loss to the plastic accounts for <5%of the observed precipitation, to siliconized plastic <1%, and bindingto Sephadex <1%. Hence, Aβ precipitates are unlikely to be adsorbing tothe Sephadex or plastic support. However, similar incubations inborosilicate glass test tubes result in 20% adsorption, which increaseto 35% in the presence of zinc (25 μM).

The glass in the Bio-Rad Econo Columns is made of 7740 Pyrex (Corning,Park Ridge, Ill.) and is composed of SiO₂, 80.6%; B₂O₃, 13.0%; Na₂O,4.0%; and Al₂O₃, 2.3%. Previous workers have found evidence linkingaluminosilicates with β-amyloid deposition (Masters et al., EMBO J.4:2757-2763 (1985a); Candy et al., Lancet 1:354-357 (1986)). In light ofthese reports, experiments were performed to further investigate thephenomena which were observed for precipitation of Aβ on 7740 Pyrexglass. Rapid and extensive binding of Aβ to kaolin, an insolublehydrated aluminum silicate was observed. Moreover, incubation of Aβ (0.4mg/ml) with Sephadex (5%, v/v) in the presence of zinc, copper, or EDTAcauses only small changes in solubility, some of which is probably dueto binding of the peptide to the plastic reaction vessels (FIG. 3 a).Incubation of Aβ (0.4 mg/ml) with kaolin (5%, v/v, 5 min, roomtemperature), causes precipitation of up to 87% of the peptide present.This precipitation is greatest in the presence of zinc (25 μM) where theamount of Aβ recovered from the zinc incubation supernatant is nearlyhalf of the amount recovered from the EDTA incubation supernatant (FIG.3 b). The effect of copper (25 μM) upon kaolin-induced Aβ precipitationis similar to the effect of EDTA (FIG. 3 b). The binding of Aβ to kaolinis not reversible to subsequent treatment with 10 mM EDTA, but can beeluted by 2 M NaOH.

To further test whether zinc induces irreversible precipitation of Aβ inthe absence of kaolin, Aβ incubated with Zn²⁺ (200 μM, 1-24 h, 20° C.)was subjected to SDS Tris/Tricine gel electrophoresis. The monomericspecies was the major band detected on Coomassie-stained gels andmigrated identically to unincubated Aβ, indicating that zinc does notinduce covalent or SDS-resistant polymerization of Aβ.

The APPα-secretase site at Lys-16 (Esch et al., Science 248:1122-1124(1990); Sisodia et al., Science 248:492495 (1990)) in Aβ is within theobligatory zinc binding region. The ability of Zn²⁺ to protect Aβ fromsecretase-type cleavage was investigated using the serine-proteasetrypsin, whose activity is unaffected by zinc. Amino-terminal sequenceon Aβ tryptic digestion products transferred to polyvinylidenedifluoride membrane following SDS-polyacrylamide gel electrophoresisindicated two detectable fragments corresponding to residues 6-40 and17-40 (FIG. 4 a). The predicted tryptic cleavage product representingresidues 29-40 did not appear on the blot and may not be retained by thepolyvinylidene difluoride membrane during transfer and treatment.Digestion is inhibited by the presence of increasing concentrations ofZn²⁺. At 200 μM, Zn²⁺ causes complete inhibition of Aβ hydrolysis;however, at this zinc level, tryptic activity is also slightlyinhibited. Probing the blot with ⁶⁵Zn²⁺ confirmed the zinc bindingidentity of the peptide fragments and facilitated quantification of thehydrolysis of the zinc binding site (FIG. 4 b). The rate of digestion ofAβ₁₋₄₀ and the Aβ₆₋₄₀ fragment is inhibited by the presence of zinc,whereas the digestion of the Aβ₁₇₋₄₀ fragment is not inhibited byincreasing zinc concentrations. Hence, only the peptides possessing theintact zinc binding domain of Aβ (residues 6-28), and therefore capableof binding Zn²⁺ (FIG. 4 b), have their rates of digestion inhibited byzinc in this experiment. These data indicate that secretase-typecleavage of Aβ can be inhibited by Zn²⁺ binding to the Aβ substrate. Theresults of the preceding experiments can be summarized as follows.

Firstly, the data indicates that soluble Aβ₁₋₄₀ possesses high and lowaffinity zinc binding sites. Secondly, the zinc binding site on Aβ mapsto residues 6-28, with possibly conformational- and histidine-dependentproperties. Thirdly, the affinity constants for zinc binding indicatethat both binding associations are within physiological zincconcentrations. The binding of zinc may inhibit the action ofα-secretase type cleavage of the peptide. Furthermore, occupancy of thelow affinity binding site may be associated with acceleratedprecipitation of Aβ by aluminum silicate (kaolin). Occupancy of the highaffinity site appears to have little effect on Aβ precipitation and isvery highly specific, although the data cannot exclude the possibilityof specific binding sites for alternative metals elsewhere on Aβ.Finally, copper's strong conformational interaction (dimerization andfluorescence) with Aβ indicates that it may also directly interact withthe peptide and may have a role in preventing Aβ precipitation ontoaluminum silicate.

Extracellular zinc may modify the adhesiveness of APP to extracellularmatrix elements (Bush et al., J. Biol. Chem. 268:16109-16112 (1993)) andthus be an important factor in the physiology of the protein. Althoughthe physiological function of APP remains unclear, the protein isthought to play a role in cell adhesiveness (Shivers et al., EMBO J.7:1365-1370 (1988)) and neurite outgrowth (Milward et al., Neuron9:129-137 (1992)). Physiological function of the Aβ-zinc interaction isalso unclear, however, increased resistance of Aβ to proteolyticcleavage in the presence of zinc would increase the peptide's biologicalhalf-life, and the resulting increase in adhesiveness may also promoteits binding to extracellular matrix elements. It has been reportedrecently that Aβ also promotes neurite outgrowth by complexing withlaminin and fibronectin in the extracellular matrix (Koo et al., Proc.Natl. Acad. Sci. USA 90:4748-4752 (1993)). Hence, both APP and Aβ mayinteract with the extracellular matrix to modulate cell adhesion. Thepossibility that zinc is a local environmental cofactor modulating thisinteraction merits further investigation.

APP is abundant in platelets and brain (Bush et al., J. Biol. Chem. 265:15977-15983 (1990)) where zinc is also highly concentrated (Baker etal., Thromb. Haemostasis 39:360-365 (1978); Frederickson, C. J., Int.Rev. Neurobiol. 31:145-328 (1989)). Although APP is concentrated invesicles in both of these tissues (Bush et al., J. Biol. Chem.265:15977-15983 (1990); Schubert et al., Brain Res. 563:184-194 (1991)),and zinc is actively taken up (Wolf et al., Neurosci. Lett. 51:277-280(1984)) and stored in synaptic vesicles in nerve terminals throughoutthe telencephalon (Perez-Clausell and Danscher, Brain Res. 3371:91-98(1985), the colocalization of APP with zinc in these vesicles has yet tobe demonstrated. Vesicular zinc storage is thought to play a role instabilizing functional molecules such as nerve growth factor (NGF) andinsulin as insoluble intravesicular precipitates (Frederickson et al.,J. Histochem. Cytochem. 35:579-583 (1987)). Zinc may similarly play arole in stabilizing APP and Aβ.

The well characterized interaction between insulin and zinc has severalstriking parallels to the interaction of Aβ and zinc. Like Aβ insulinexhibits histidine-dependent high-affinity (K_(a)=5 μM) and low-affinity(K_(a)=140 μM) zinc binding with stoichiometries of 1:1 (insulin:zinc)and 1:2, respectively (Goldman and Carpenter, Biochemistry 13:4566-4574(1974)). Additionally, metal-free insulin exhibits a pH-dependentpolymerization pattern consisting of monomer, dimer, tetramer, hexamer,and higher aggregation states, in dynamic equilibrium. At neutral pH,zinc and other divalent metal ions shift the equilibrium toward thehigher aggregation states. At stoichiometric ratios of Zn²⁺:insulin inexcess of 0.33, the peptide precipitates (Fredericq, E., Arch. Biochem.Biophys. 65:218-228 (1956)), reminiscent of zinc's effects upon Aβobserved in the current studies.

Aβ chelates zinc with such high affinity that reports of its neurotoxiceffects in neuronal cultures (Yankner et al., Science 250:279-282(1990); Koh et al., Brain Res. 533:315-320 (1990)) might be explained bya disturbance of zinc homeostasis. Aβ accumulates most consistently inthe hippocampus, where extreme fluctuations of zinc concentrations occur(0.15-300 μM) (Frederickson, C. J., Int. Rev. Neurobiol. 31:145-328(1989)), e.g., during synaptic transmission (Assaf and Chung, Nature308:734-736 (1984; Howell et al., Nature 308:736-738 (1984); Xie andSmart, Nature 349:521-524 (1991)). Choi and co-workers (Weiss et al.,Nature 338:212 (1989)) have proposed that this trans-synaptic movementof zinc may have a normal signaling function and may be involved in longterm potentiation. The hippocampus is the region of the brain that bothcontains the highest zinc concentrations (Frederickson et al., BrainRes. 273:335-339 (1983)) and is most severely and consistently affectedby the pathological lesions of Alzheimer's disease (Hyman et al., Ann.Neurol. 20:472-481 (1986)). One of the prominent neurochemical deficitsin Alzheimer's disease is cholinergic deafferentation of thehippocampus, which has been shown to raise the concentration of zinc inthis region (Stewart et al., Brain Res. 290:43-51 (1984)).

The rapid zinc-accelerated precipitation of Aβ by aluminum silicate(kaolin) is significant because of the candidacy of aluminum as apathogenic agent in AD (Perl and Brody, Science 208:297-299 (1980)).Recent reports of Zn²⁺- and Al³⁺-induced sedimentation of Aβ (Mantyh etal., J. Neurochem. 61:1171-1174 (1993)), and the nucleation of Aβprecipitation by aluminosilicate (Candy et al., Biochem. Soc. Trans.21:53S (Abstract) (1992)) also support these observations.

Evidence for altered zinc metabolism in AD includes decreased temporallobe zinc levels (Wenstrup et al., Brain Res. 533:125-131 (1990);Constantinidis, Encephale 16:231-239 (1990); Corrigan et al., Biometals6:149-154 (1993)), elevated (80%) cerebrospinal fluid levels (Hershey etal., Neurology 33:1350-1353 (1983)), increased hepatic zinc with reducedzinc bound to metallothionein (Lui et al., J. Am. Geriatr. Soc.38:633-639 (1990)), a Zn²⁺-modulated abnormality of APP in AD plasma(Bush et al., Ann. Neurol. 32:57-65 (1992)), an increase inextracellular Zn²⁺-metalloproteinase activities in AD hippocampus(Backstrom et al., J. Neurochem. 58:983-992 (1992)), and decreasedlevels of astrocytic growth inhibitory factor, a metallothionein-likeprotein which chelates zinc (Uchida et al., Neuron 7:337-347 (1991)).Collectively, these reports indicate that there may be an abnormality inthe uptake or distribution of zinc in the AD brain causing highextracellular concentrations and low intracellular concentrations.Meanwhile, environmentally induced elevations of brain concentrations ofboth zinc (Duncan et al., J. Neurosci. 12:1523-1537 (1992)) and aluminum(Garruto et al., Proc. Natl. Acad. Sci. USA 81:1875-1879 (1984); Perl etal., Science 217:1053-1055 (1982)) have been implicated in thepathogenesis of GALS/PDC complex, a disease also characterized byneurofibrillary tangles (Guiroy et al., Proc. Natl. Acad. Sci. USA84:2073-2077 (1987)). Interestingly, a pervasive abnormality of zincmetabolism manifested by immunological and endocrine dysfunction hasbeen described as a common complication of Down's syndrome (Franceschiet al., J. Ment. Defic. Res. 32:169-181 (1988); Bjorksten et al., Acta.Pediatr. Scand. 69:183-187 (1980)), a condition characterized by theinvariable onset of presenile Aβ deposition and Alzheimer's disease(Rumble et al., N. Engl. J. Med. 320:1446-1452 (1989)).

These results indicate that abnormally high zinc concentrations increaseAβ resistance to secretase-type cleavage and also accelerate Aβprecipitation onto aluminosilicates. Zinc-induced accumulation of Aβ inthe neuropil may, in turn, invoke a glial inflammatory response, freeradical attack, and oxidative cross-linking to form an, ultimately,“mature” amyloid. Collectively, these findings support the biochemicalrationale for the chelation approach in the therapy of Alzheimer'sdisease (Crapper McLachlan et al., Lancet 337:1304-1308 (1991)), sincereduction of cerebral concentrations of both aluminum and zinc couldpotentially decelerate the precipitation of Aβ. The assay of the presentinvention is ideally suited for the preparation of a kit. Such a kit maycomprise a carrier means being compartmentalized to receive in closeconfinement therein one or more container means, such as vials, tubes,and the like, each of said container means comprising one of theseparate elements of the assay to be used in the method. For example,there may be provided a container means containing standard solutions ofthe Aβ peptide or lyophilized Aβ peptide and a container meanscontaining a standard solution or varying amounts of a heavy metalcation capable of binding to the peptide comprising at least amino acids6 to 28 of Aβ peptide, in any form, i.e., in solution or dried, solubleor insoluble, in addition to further carrier means containing varyingamounts and/or concentrations of reagents used in the present methods,e.g., standard solutions or varying amounts of chealators of heavy metalcations in any form, in solution or dried. Standard solutions of Aβpeptide preferably have concentrations above about 10 μM, morepreferably from about 10 to about 25 μM or if the peptide is provided inits lyophilized form, it is provided in an amount which can besolubilized to said concentrations by adding an aqueous buffer orphysiological solution. Standard solutions of heavy metal cationspreferably have concentrations above 300 nM, more preferably about 25μM. The standard solutions of analytes may be used to prepare controland test reaction mixtures for comparison, according to the methods ofthe present invention for determining whether a compound inhibitsformation of Aβ amyloid.

These studies show that Aβ binds zinc in a saturable and specificmanner. Moreover, they demonstrate that physiological concentrations ofZn²⁺ increase the resistance of the peptide to proteolytic catabolismand promote Aβ precipitation by aluminosilicate. Based on thesefindings, it is possible that excessive zinc concentrations accelerateAβ deposition in AD and related pathological conditions.

Further, the effects of physiological concentrations of zinc upon thestability of synthetic human Aβ₁₋₄₀ in solution were studied, using therat/mouse species of the peptide (“rat Aβ”) for comparison. SolubleAβ₁₋₄₀ is produced by rat neuronal tissue (C. Haass and D. J. Selkoe,personal communication), however, Aβ amyloid deposition is not a featureof aged rat brains (D. W. Vaughan and A. Peters, J. Neuropathol. Exp.Neurol. 40:472 (1981)). β-amyloidogenesis occurs in other aged mammalspossessing the human Aβ sequence, which is strongly conserved in allreported animal species, except rat and mouse (E. M. Johnstone, M. O.Chaney, F. H. Norris, R. Pascual, S. P. Little, Mol. Brain Res. 10:299(1991)). The rat/mouse Aβ substitutions (Arg→Gly, Tyr→Phe and His→Arg atpositions 5, 10 and 13, respectively [B. D. Shivers et al., EMBO J.7:1365 (1988)]) appear to cause a specific change in the peptide'sphysicochemical properties sufficient to confer upon the peptide itsrelative immunity to amyloid formation. Since zinc binding to humanAβ₁₋₄₀ is histidine-mediated, the altered zinc binding properties of ratAβ are entirely consistent with the proposed mechanism and binding siteof the human peptide.

The binding affinity of zinc to rat Aβ₁₋₄₀ was studied in a ⁶⁵Zncompetitive assay system as described in Example 1 (FIG. 1), to measurethe K_(A) of zinc binding to human Aβ₁₋₄₀. In contrast to human Aβ₁₋₄₀,the Scatchard analysis of zinc binding to rat Aβ₁₋₄₀ reveals only onebinding association (K_(A)=3.8 μM), with 1:1 stoichiometry (FIG. 5).

It was observed that the recovery of human Aβ₁₋₄₀ in filtrationchromatography is dramatically reduced in the presence of zinc, due, inpart, to increased adhesiveness of Aβ. To determine whether theaggregation of human Aβ₁₋₄₀ is also enhanced in the presence of zinc,the peptide was incubated with various concentrations for 30 minuteswith Zn²⁺ (25 μM) or EDTA and then filtered the solutions through 0.2μfilters. Zinc caused up to 80% of the available peptide to aggregateinto >0.2μ particles (FIG. 6A). (Incubation of Aβ₁₋₄₀ solutions in thefilter devices, without actual filtration, indicated that there was nonon-specific loss of peptide to the plastic or membrane surfaces.) Thereappears to be a shallow negative log-linear relationship between humanAβ peptide concentration and the proportion of filterable peptide in 25μM Zn²⁺, but even at the lowest concentration tested (0.8 μM), >70% ofthe human Aβ₁₋₄₀ solution aggregated. In contrast, the effect of Zn²⁺ onrat Aβ₁₋₄₀ was unremarkable, with no aggregation of a 0.8 μM peptidesolution detected under the same conditions, and only 25% aggregation ofa 4 μM solution. Meanwhile, in the presence of EDTA, human and ratAβ₁₋₄₀ solutions behaved indistinguishably, with no detectableaggregation observed at 0.8 μM, and ≈15% aggregation at higher peptideconcentrations.

Next, the formation of >0.2μ Aβ particles was titrated againstincreasing zinc concentrations (FIG. 6B), and a shallow response curvefor human Aβ₁₋₄₀ (1.6 μM) was observed until the zinc concentrationreached 300 nM, corresponding to the saturation of high-affinitybinding. At zinc concentrations above 300 nM, corresponding tolow-affinity binding, human Aβ₁₋₄₀ dramatically aggregates. In contrast,rat Aβ₁₋₄₀ remains stable in the presence of up to 10 μM zinc, and onlyat 25 μM zinc was aggregation observed.

To determine the effects of zinc on Aβ₁₋₄₀ at physiological peptideconcentrations requires an assay more sensitive than spectroscopy.(Human Aβ₁₋₄₀ at 0.8 μM in buffer 1 corresponds to 0.090 absorbanceunits at 214 mm. Aggregation studies of peptides at lower startingconcentrations would involve readings at the limits of sensitivity).Thus, the effects of zinc on ¹²⁵I-human Aβ₁₋₄₀ used as a tracer in thepresence of unlabeled peptide was characterized. Unlike its unlabeledprecursor, ¹²⁵I-Aβ₁₋₄₀ (at 1.6 μM total peptide) remained stable in thepresence of increasing zinc concentrations, indicating that ¹²⁵I-Aβ₁₋₄₀is not a suitable tracer (FIG. 6B). The tracer is iodinated on thetyrosine residue at position 10, which is a phenylalanine in the ratpeptide. Thus, the tyrosine residue may be critical to the stability ofthe human peptide. These data may also explain why a recent reportrequired relatively high concentrations of Zn²⁺ (1 mM) to precipitate¹²⁵I-human Aβ₁₋₄₀ in centrifugation studies (P. W. Mantyh et al., J.Neurochem. 61:1171 (1993)). Extrapolating the curve in FIG. 6A to 0.6 nMcurrently provides the best estimate of the effect of zinc uponphysiological Aβ concentrations (M. Shoji et al., Science 258:126(1992); P. Seubert et al., Nature 359:325 (1992)), and indicates that25% of the peptide would aggregate into >0.2μ particles under theseconditions. The specific vulnerability of human Aβ₁₋₄₀ for Zn²⁺ isindicated by the observation that Zn²⁺ is the only one of several metalions tested on an equimolar basis, including Al³⁺, to induce significantaggregation of human Aβ₁₋₄₀ in this system (FIG. 6C).

Next, the kinetics of the assembly of zinc-induced human Aβ₁₋₄₀aggregates (FIG. 6D) was investigated. (In order to achieve time pointmeasurements of less than 1 minute, the procedure was modified so thatsamples were centrifuged at 2500 g, allowing the sample volume to becompletely filtered in 40 seconds.) The data obtained indicate thatfollowing the addition of stock Aβ₁₋₄₀ in water (15.9 μM, pH 5.6) toZn²⁺ (25 μM) in saline buffer (pH 7.4) there is a near-instantaneousaggregation of the peptide (1.6 μM final concentration) into filterableparticles with two phases observed over two hours. The initial phase israpid, with a half-maximal assembly rate of ≈0.4 μM/min. The steadystate of the second phase is achieved within about 2 minutes, whereuponparticle assembly proceeds at a rate of 3.2 nM/min with no evidence ofsaturation within 2 hours. At this rate, the available peptide isexhausted within five hours of initiation. Although the addition of EDTAbuffer maused the near-instantaneous aggregation of 20% of the 1.6 μMAβ₁₋₄₀ solution into >0.2μ particles, no further particle assembly wasobserved over the time course of the experiment. In comparison, humanAβ₁₋₄₀ (20 μM in PBS, pH 7.4) has been reported to be stable for 10 days(J. T. Jarrett, E. P. Berger, P. T. Lansbury, Biochemistry 32:4693(1993)), and seeding the solution with Aβ₁₋₄₂ (2 μM), the moreamyloidogenic Aβ species, induced aggregation of this solution which washalf-maximal only after 4-5 days. Thus, the results presented hererepresent a major advance among attempts to induce amyloid formation invitro using the wild-type form of the main species of secreted Aβ(Aβ₁₋₄₀).

To estimate the size of the Aβ aggregates formed in the presence ofzinc, Aβ₁₋₄₀ (1.6 μM) was incubated with Zn²⁺ (25 μM) or EDTA and thenpassed through filters with various pore sizes (FIGS. 7 a and 7 b).Following incubation in EDTA, human Aβ₁₋₄₀ assembled into populations ofheterogeneous particle sizes, >0.1/μ: 47%, >0.22μ: 40%, >0.65μ: 32%. Thecomparable proportions of filtered rat Aβ₁₋₄₀ particles were, >0.1μ:36%, >0.22μ: 27%, >0.65μ: 25%. Upon incubation with Zn²⁺ (25 μM), theproportion of >0.65μ rat peptide particles increased only slightly,however the proportion of >0.65μ human peptide particles dramaticallyincreased, recruiting 82% of the available peptide. Interestingly, theproportions of >0.1μ and >0.22μ particles formed from the human Aβ₁₋₄₀also increased by 50 and 55%, respectively, following incubation withZn²⁺, however, the same reaction induced only a 20% and 30% increase,respectively, in the amounts of these particles assembled from ratpeptide. Remarkably, only 4% of the human Aβ₁₋₄₀ incubated with Zn²⁺remained in solution following 0.1μ filtration. Collectively, these dataindicate that the human species of Aβ₁₋₄₀ differs from the rat speciesboth in the extent and size of zinc-induced particle formation.

The stoichiometry of zinc:human Aβ in these aggregates is at least 1:1(FIG. 7 c), but increases to 1.3:1 with the smaller (0.1μ) pore sizefilters. Because the stoichiometries for high- and low-affinity Zn:Aβbinding are ≈1:1 and ≈2:1 respectively, these data indicate thatformation of >0.65μ Aβ aggregates is mediated by high-affinity zincinteraction, whereas low-affinity zinc interaction most likelycontributes to the formation of smaller (<0.22μ) aggregates.Interestingly, when the retained aggregates are washed with EDTA, only22% of the peptide is recovered from >0.65 aggregates, although thecomplexed zinc (using ⁶⁵Zn as tracer) is completely recovered (FIG. 7d). This indicates that zinc-induced Aβ aggregation is largelyirreversible by chelation. The amount of ≦0.22μ peptide resolubilized byEDTA treatment is 7% greater, which may reflect the increasedcontribution of low-affinity zinc binding to the smaller,chelation-reversible, Aβ particle formation.

Sedimentation of zinc-induced Aβ particles by centrifugation resulted inan abundant precipitate of human Aβ₁₋₄₀ which stained with Congo Red(FIG. 8 a) and manifested green birefringence under polarized light(FIG. 8 b), meeting the criteria for tinctorial amyloid formation.However, following incubation with Zn²⁺ under the same conditions, therat peptide formed significantly fewer and smaller particles, withminimal birefringence. No rat Aβ amyloid was induced by Zn²⁺concentrations of less than 10 μM, whereas, by tinctorial criteria,human Aβ amyloid was induced by Zn²⁺ concentrations as low as 3 μM. Inneither case was Congo Red-stained material detected followingincubation with EDTA-containing buffer.

Taken together, these data indicate that soluble human Aβ₁₋₄₀ has adramatically greater propensity than rat Aβ₁₋₄₀ to form amyloid in thepresence of physiological zinc concentrations. The tinctorial amyloidaggregates are frequently as large as the amorphous amyloid plaque corespurified from AD brain tissue (C. L. Masters et al., Proc. Natl. Acad.Sci. USA 82:4245 (1985)). Meanwhile, the small degree (10-20%) of >0.2μAβ₁₋₄₀ particle assembly observed following the incubation of Aβ₁₋₄₀with EDTA probably reflects the relatively slow aggregation which occursin the presence of neutral pH (S. Tomski and R. M. Murphy, Arch.Biochem. Biophys. 294:630 (1992)) and NaCl (C. Hilbich, B.Kisters-Woike, J. Reed, C. L. Masters, K. Beyreuther, J. Mol. Biol.218:149 (1991)). Hence, the specific vulnerability of human Aβ tozinc-induced amyloid formation is a promising explanation for aspects ofthe pathology of AD and related pathological conditions.

The cerebral cortex, and especially the hippocampus, contains thehighest concentrations of zinc in the body (C. J. Frederickson, M. A.Klitenick, W. I. Manton, J. B. Kirkpatrick, Brain Res. 273:335 (1983)),and is exposed to extreme fluctuations of extracellular zinc levels(0.15 to 300 μM, C. J. Frederickson, Int. Rev. Neurobiol. 31:145(1989)), e.g. during synaptic transmission (S. Y. Assaf and S.-H. Chung,Nature 308:734 (1984); G. A. Howell, M. G. Welch, C. J. Frederickson,Nature 308:736 (1984)). The cortical vasculature contains anintraluminal zinc concentration of 20 μM (I. J. T. Davies, M. Musa, T.L. Dormandy, J. Clin. Pathol. 21:359 (1968)), but the perivascularinterstitial zinc concentration is 0.15/μM (C. J. Frederickson, Int.Rev. Neurobiol. 31:145 (1989)). Both sites of high zinc concentrationgradients are severely and consistently affected by the pathologicallesions of AD (B. T. Hyman, G. W. Van Hoesen, L. J. Kroner, A. R.Damasio, Ann. Neurol. 20:472 (1986); G. G. Glenner and C. W. Wong,Biochem. Biophys. Res. Commun. 120:885 (1984)). Interestingly, aprominent neurochemical deficit in AD is cholinergic deafferentation ofthe hippocampus, which raises the concentration of zinc in this region(G. R. Stewart, C. J. Frederickson, G. A. Howell, F. H. Gage, Brain Res.290:43 (1984)). Additional evidence for altered cerebral zinc metabolismin AD include decreased temporal lobe zinc levels (D. Wenstrup, W. D.Ehmann, W. R. Markesbery, Brain Res. 533:125 (1990); J. Constantinidis,Encephale 16:231 (1990); F. M. Corrigan, G. P. Reynolds, N. I. Ward,Biometals 6:149 (1993)), elevated (80%) CSF levels (C. O. Hershey et al,Neurology 33:1350 (1983)), an increase in extracellularZn²⁺-metalloproteinase activities in AD hippocampus (J. R. Backstrom, C.A. Miller, Z. A. Tökés, J. Neurochem. 58:983 (1992)), and decreasedlevels of astrocytic growth-inhibitory factor, a metallothionein-likeprotein which chelates zinc (Y. Uchida, K. Takio, K. Titani, Y. Ihara,M. Tomonaga, Neuron 7:337 (1991)). Recently, a clinical study assayedthe effects of oral zinc supplementation (6.7-fold the recommended dailyallowance, a dose commonly found in nutritional supplements) uponcognition and plasma APP levels in AD subjects and age-matched controls.Five sequentially-studied AD subjects each experienced an acute declinein cognition within forty-eight hours of ingesting the zinc dose. Underthe same conditions, age-matched control subjects remained unaffected bythe dose. Among the abnormal changes of neuropsychological measurementstaken of the AD group was a 31% drop in Mini-Mental State Examination(M. F. Folstein, S. E. Folstein, P. R. McHugh, J. Psychiatr. Res. 12:189(1975)) scores, after four days of zinc supplementation. Thisrepresented a deterioration which, in the ordinary course of thedisease, would only be expected after two to four years (Galasko et al.,JAGS 39:932 (1991)). Plasma APP levels also rose significantly inresponse to zinc in both the AD and the control groups. All changes wererapidly reversible following cessation of the four day supplementation.Collectively, these reports indicate that there may be an abnormality inthe uptake or distribution of zinc in the AD brain. Pervasiveabnormalities of zinc metabolism, and premature AD pathology, are alsocommon clinical complications of Down's syndrome (C. Franceschi et al.,J. Ment. Defic. Res. 32:169 (1988); B. Rumble et al., N. Engl. J. Med.320:1446 (1989)).

The data presented here indicate that stability in the presence ofphysiological concentrations of zinc clearly differentiates thepropensity of human and rat Aβ₁₋₄₀ peptide species to form amyloid. Therapid induction of tinctorial human Aβ amyloid, under physiologicallyrelevant conditions, at peptide concentrations more than an order ofmagnitude lower than the lowest levels achieved previously for Aβ₁₋₄₀aggregation, and within two minutes of incubation, establishes a novelassay system for the study of Aβ amyloidosis. More importantly, thesefindings can have profound implications for the potential role of zincin Alzheimer-associated neuropathogenesis.

The following examples are provided by way of illustration to furtherdescribe certain preferred embodiments of the invention, and are notintended to be limiting of the present invention, unless specified.

EXAMPLES Experimental Procedures

Unless otherwise indicated, the following experimental procedures,materials, and reagents were used in the present invention:

Reagents—Precautions taken to avoid zinc contamination included usinganalytical-grade reagents, electrophoresis-grade Tris-HCl (Bio-Rad), andhighly deionized water. Aβ₁₋₁₇ was synthesized by the BiopolymersLaboratory, MIT. Aβ₄₀₋₁ (reverse peptide) was purchased from Bachem(Torrance, Calif.). Other reagents were from Sigma. Aβ₁₋₄₀ and Aβ₁₋₂₈results were replicated with peptides from Bachem and Sigma. Aβ₁₋₄₀results were also replicable with peptide synthesized by W. M. KeckFoundation Biotechnology Resource Laboratory, Yale University. ⁶⁵Zn waspurchased from Amersham Corp.

⁶⁵Zn²⁺ Binding Studies—Dissolved peptides (1.2 nMol, unless other wisestated) were dot-blotted onto polyvinylidene difluoride membrane (0.2-μmpore size; Pierce Chemical Co.), washed twice with chelating buffer (200μl×100 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, pH 7.4), then five times withblocking buffer (200 μl×100 mM NaCl, 20 mM Tris-HCl, 1 mM MnCl₂, pH7.4), and then incubated (60 min, 20° C.) with ⁶⁵Zn (unless otherwisestated 130,000 cpm, 74 mM ⁶⁵ZnCl₂ in 200 μl of blocking buffer±competingmetal ion chloride). The dot-blot was then washed with blocking buffer(5×200 μl), the dot excised, placed in a test tube, and assayed byγ-counting (11% efficiency). The equilibration volume for stoichiometryestimates was regarded as 6×200 μl. The 214 nm UV absorbance of theunbound flow-through was assayed to determine the total amount ofpeptide remaining bound onto the membrane. Peptide stock concentrationswere confirmed by amino acid analysis. To alter the pH, the ⁶⁵Znincubation was carried out in the presence of 100 mM buffer: MOPS (pH6.5-7.0), MES (pH 5.0-6.0), acetate (pH 3.5-4.5). The dot-blot apparatuswas washed with detergent and EDTA (50 mM) then rinsed and siliconizedbetween use.

Aβ Chromatography—Aβ (55 μg) was incubated with metal salt solution orEDTA in siliconized 1.5-ml plastic reaction vessels in 100 mM NaCl, 20mM Tris-HCl, pH 7.4 (“TBS,” 100 μl, 1 h, 37° C.). Aβ was stored inaliquots of 0.52 mg/ml in water at −20° C., then kept at 4° C. whenthawed. Reagents were mixed without vortex mixing. The incubated Aβ wasdirectly applied to a G50 SF (Pharmacia, Uppsala, Sweden) column(Bio-Rad Econo-Column, 30×0.7 cm) pre-equilibrated with metal saltsolution or EDTA (50 μM) in TBS at 20° C. and eluted at 8 ml/h (Wizperistaltic pump, Isco, Lincoln, Nebr.). Absorbance was measured at 254and 214 nm (Type 6 optical unit, Isco). The amount of Aβ eluting atvarious peaks was estimated from the area under the curve. This waspossible because the relationship of UV absorbance was determined to belinear over the range of Aβ dilutions used in these studies, indicatingthat absorbance is proportional to the amount of peptide present despitepolymerization state (see below). The maximum recovery of Aβ occurs inthe presence of EDTA. Because the sample eluted in a volume ofapproximately 15 ml, the average concentration of the peptide on thecolumn was 0.8 μM.

To study the effects of protein blocking upon adsorption of β to thechromatography column, a Sephadex G50 SF column which had beencharacterized previously for Aβ behavior was eluted with 3% bovine serumalbumin (BSA) in TBS (50 ml) and equilibrated with non-BSA-containingbuffer, subsequent to repeating the Aβ experiments.

Spectroscopic Assay—Measurements were performed on a Hewlett-Packard8452A diode array spectrophotometer using a 1-cm path length quartzcuvette. Concentration versus absorbance curves were performed at 214nm, 254 nm, 280 nm, and full spectrum. 214 nm readings were 50-fold moresensitive in detecting the peptide than 254 nm readings, whereas the 280nm readings of low micromolar Aβ solutions were below sensitivity limitsand hence could not be used in these studies. The standard curvesgenerated were linear at concentrations below 0.1 mg/ml. In addition,the effects of Cu²⁺, Zn²⁺, EDTA, and TBS upon absorbance were examined.At concentrations below 0.1 mg/ml, adjusting the peptide in water to TBScaused ≈15% quenching. Cu²⁺-, Zn²⁺-, and EDTA-containing Aβ solutionswere studied for artifactual absorbance over the linear range of the 214nm absorbance curve. 1 mM EDTA caused 60% quenching, hence 50 μM EDTAwas employed, contributing a similar degree of quenching to thatobserved with Cu²⁺ and Zn²⁺.

Aβ Binding to Kaolin (Aluminum Silicate)—Kaolin suspension was preparedin high performance liquid chromatography water (Fisher), defined, andadjusted to 50% (v/v). Aβ (40 μg) was incubated in siliconized reactionvessels with either kaolin or Sephadex G50 SF (10 μl×50% (v/v)) in Cu²⁺,Zn²⁺, or EDTA (100 μl in TBS, 5 min, room temperature). The suspensionwas then pelleted (1500×g, 3 min) and the supernatant removed anddiluted 20-fold with water to bring the UV absorbance readings into thelinear range. Samples were assayed at 214 nm before and after incubationwith kaolin or Sephadex.

Tryptic Digestion of Aβ-Aβ₁₋₄₀ (13.9 μg) was incubated with Zn²⁺ (12 μlin blocking buffer, 1 h, 37° C.) and then digested with trypsin (12 ng,3 h, 37° C.). The reaction was stopped by adding SDS sample buffercontaining phenylmethylsulfonyl fluoride (1 mM), boiling the samples (5min), and applying the samples to Tris/Tricine gel electrophoresis andtransfer. The blot was washed with EDTA, Coomassie-stained, incubatedwith ⁶⁵Zn²⁺, individual bands were excised, assayed for ⁶⁵Zn²⁺ binding,and N-terminal sequenced to confirm the identity of the digestionproducts. The effects of Zn²⁺ (up to 100 μM in TBS) on the activity oftrypsin, itself, were assayed by assay of Z-Arg-amido-4-methylcoumarin(Sigma) fluorescent cleavage product and determined to be negligible. Itwas found that 200 μM Zn²⁺, however, inhibited tryptic activity by 12%.

Zinc- or Copper-treated Microwell Plate

Any standard microtitre plate, for example a Costar catalog no. 9017,can be used for making the heavy metal cation substrate which can trapAβ protein. The plate is coated with a solvated nitrilotriacetic acid.Next, the divalent metal ion of choice, for example, zinc or coppermetal cation, is added, which complexes to the surface of the plate. Apreferred metal cation microtiter plate is available from Xenopore,Saddle Brook, N.J. (catalog number for zinc plates: ZCP00100, catalognumber for copper plates: CCP00100). It is preferred that like the zincand copper plates made by Xenopore, there be at least two freecoordination sites available for binding to Aβ protein. In this way, theAβ protein can competitively attach to and stay bound to the substratevia the heavy metal cation.

Chelating-Sepharose Chromatography

The chelating-Sepharose resin (250 μl) was poured into a disposablepolystyrene column (Pierce, 29920) and packed between two porouspolyethylene discs. In the following steps solutions were allowed todrain through the gel bed by gravity. The gel was first pre-equilibratedwith 5 ml of equilibration buffer (MES 50 mM, pH 5.0 and NaCl 500 mM).The sample (10-15 ml) was then loaded on to the column. The gel waswashed with 5 ml of equilibration buffer before bound protein wasrecovered by applying 1 ml of elution buffer to the gel (EDTA 50 mM, MES50 mM, pH 5.0 and NaCl 500 mM) and collecting the eluate.

Example 1 Analyses of ⁶⁵Zn²⁺ Binding to Aβ

Aliquots of Aβ were incubated (60 min) with ⁶⁵Zn²⁺ in the presence ofvarying concentrations of unlabeled Zn²⁺ (0.01-50 μM total). Theproportion of ⁶⁵Zn³⁰ binding to immobilized peptide (1.0 nmol) describedtwo binding curves as shown in FIG. 1 a (Scatchard plot). Values shownare means±S.D., n≧3. The high-affinity binding curve has been correctedby subtracting the low-affinity component, and the low-affinity curvehas had the high-affinity component subtracted. (FIG. 1 b) depictsspecificity of the Zn²⁺ binding site for various metals. Aβ wasincubated (60 min) with ⁶⁵ Zn²⁺ (157 nM, 138,000 cpm) and competingunlabeled metal ions (50 μM total). (FIG. 1 c) depicts ⁶⁵Zn²⁺ (74 nM,104,000 cpm) binding to negative (aprotinin, insulin α-chain, reversepeptide 40-1) and positive (bovine serum albumin (BSA)) control proteinsand Aβ fragments (identified by their residue numbers within the Aβsequence, gln11 refers to Aβ₁₋₂₈ where residue 11 is glutamine). Percentbinding of total counts ⁶⁵Zn²⁺/min added is corrected for the amounts(in nanomoles) of peptides adhering to the membrane. (FIG. 1 d) depictsas for 1 a, except with Aβ₁₋₂₈ peptide substituting for Aβ₁₋₄₀. 157 nM⁶⁵Zn (138,000 cpm) is used in this experiment to probe immobilizedpeptide (1.6 nmol). (FIG. 1 e) depicts pH dependence of ⁶⁵Zn²⁺ bindingto Aβ₁₋₄₀.

Example 2 Effect of Zn²⁺ and Other Metals on Aβ Polymerization Using G50Gel Filtration Chromatography

Results shown are indicative of n≧3 experiments where 55 μg of Aβ isapplied to the column and eluted in 15 ml, monitored by 254 nmabsorbance. (FIG. 2 a) depicts chromatogram of Aβ in the presence ofEDTA, 50 μM, Zn²⁺, 0.4 μM; Zn²⁺, 25 μM; and Cu²⁺, 25 μM. The elutionpoints of molecular mass standards and relative assignments of Aβ peakelutions are indicated. Mass standards were blue dextran (2×10⁶ kDa,V₀=void volume), BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c(12.4 kDa), and aprotinin (6.5 kDa). The mass of Aβ is 4.3 kDa. (FIG. 2b) depicts relative amounts (estimated from areas under the curve) ofsoluble Aβ eluted as monomer, dimer, or polymer in the presence ofvarious metal ions (25 μM), varying concentrations of Zn²⁺ or Cu²⁺ (thelikelihood of Tris chelation is indicated by upper limit estimates), andEDTA. Data for experiments performed in the presence of copper weretaken from 214 nm readings and corrected for comparison. (FIG. 2 c)depicts effects of pre-blocking the chromatography column with BSA uponthe recovery of Aβ species in the presence of zinc (25 μM), copper (25μM), or chelator.

Example 3 Aβ Binding to Kaolin (Aluminum Silicate): Effects of Zinc (25μM), Copper (25 μM), and EDTA (50 μM)

(FIG. 3 a) depicts concentration (by 214 nm absorbance) of Aβ remainingin supernatant after incubation with 10 mg of G50 Sephadex. (FIG. 3 b)depicts concentration (by 214 nm absorbance) of Aβ remaining insupernatant after incubation with 10 mg of kaolin, expressed as percentof the starting absorbance.

Example 4 Effect of Zn²⁺ Upon Aβ Resistance to Tryptic Digestion

(FIG. 4 a) depicts a blot of tryptic digests of Aβ (13.9 μg) afterincubation with increasing concentrations of zinc (lane labels, inmicromolar), stained by Coomassie Blue. Digestion products of 3.6 kDa(Aβ₆₋₄₀), and 2.1 kDa (Aβ₁₇₋₄₀), as well as undigested Aβ₁₋₄₀ (4.3 kDa),are indicated on the left. The migration of the low molecular sizemarkers (STD) are indicated (in kilodaltons) on the right. (FIG. 4 b)depicts ⁶⁵Zn²⁺ binding to Aβ tryptic digestion products. The blot in 4 awas incubated with ⁶⁵Zn²⁺, the visible bands excised, and the boundcounts for each band determined. These data are typical of n=3replicated experiments.

Example 5 Scatchard Analysis of ⁶⁵Zn Binding to Rat Aβ₁₋₄₀

Dissolved peptides (1.2 nmol) were dot-blotted onto 0.20μ PVDF membrane(Pierce) and competition analysis performed as described in Example 1 tomeasure the K_(A) of zinc binding to human Aβ₁₋₄₀ (FIG. 1).

In the present invention, rat Aβ₁₋₄₀ and human Aβ₁₋₄₀ were synthesizedby solid-phase Fmoc chemistry. Purification by reverse-phase HPLC andamino acid sequencing confined the synthesis. The tabulated results arepresented in FIG. 5. The regression line indicates a K_(A) of 3.8 μM.Stoichiometry of binding is 1:1. Although the data points for theScatchard curve are slightly suggestive of a biphasic curve, a biphasiciteration yields association constants of 2 and 9 μM, which does notjustify an interpretation of physiologically separate binding sites.

Example 6 Effect of Zinc Upon Human, ¹²⁵I-human and Rat Aβ₁₋₄₀Aggregation into >0.2μ Particles

Stock human and rat Aβ₁₋₄₀ peptide solutions (16 μM) in water wereprefiltered (Spin-X, Costar, 0.2μ cellulose acetate, 700 g), brought to100 mM NaCl, 20 mM Tris-HCl, pH 7.4 (buffer 1)±EDTA (50 μM) or metalchloride salts, incubated (30 minutes, 37° C.) and then filtered again(700 g, 4 minutes). The fraction of the Aβ₁₋₄₀ in the filtrate wascalculated by the ratio of the filtrate OD₂₁₄ (the response of theOD₂₁₄, titrated against human and rat Aβ₁₋₄₀ concentrations (up to 20 μMin the buffers used in these experiments), was determined to be linearrelative to the OD₂₁₄ of the unfiltered sample. The results aretabulated in FIG. 6. All data points are in triplicate, unlessindicated. (FIG. 6 a) Proportions of Aβ₁₋₄₀ incubated±Zn²⁺ (25 μM) orEDTA (50 μM) and then filtered through 0.2μ titrated against peptideconcentration. (FIG. 6 b) Proportion of Aβ₁₋₄₀ (1.6 μM) filtered through0.2μ titrated against Zn²⁺ concentration. ¹²⁵I-human Aβ₁₋₄₀ (¹²⁵I-humanAβ₁₋₄₀ was prepared according to the method in J. E. Maggio, PNAS USA89:5462-5466 (1992) (15,000 CPM, the kind gift of Dr. John Maggio,Harvard Medical School) was added to unlabeled Aβ₁₋₄₀ (1.6 μM) as atracer, incubated and filtered as described above. The CPM in thefiltrate and retained on the excised filter were measured by aγ-counter. FIG. 6 c) Proportion of Aβ₁₋₄₀ (1.6 μM) filtered through 0.2μfollowing incubation with various metal ions (3 μM). The atomic numberof the metal species is indicated. (FIG. 6 d) Effects of Zn²⁺ (25 μM) orEDTA (50 μM) upon kinetics of human Aβ₁₋₄₀ aggregation measured by 0.2μfiltration. Data points are in duplicate.

Example 7 Size Estimation of Zinc-induced Aβ Aggregates

(FIGS. 7 a and 7 b) Proportion of Aβ₁₋₄₀ (1.6 mM in buffer 1 (100 mMNaCl, 20 mM Tris-HCl, pH 7.4)), was incubated±Zn²⁺ (25 μM) or EDTA (50μM) and was then filtered through filters of indicated pore sizes(Durapore filters (Ultrafree-MC, Millipore) were used for this study,hence there is a slight discrepancy between the values obtained with the0.22μ filters in this study compared to values obtained in FIG. 2 using0.2μ Costar filters). (FIG. 7 c) ⁶⁵ZnCl₂ (130,000 CPM, 74 nM) was usedas a tracer of the assembly of the zinc-induced aggregates of humanAβ₁₋₄₀ produced in FIG. 3A. By determining the amounts of Aβ₁₋₄₀ and⁶⁵Zn in the filtrate, the quantities retarded by the filters could bedetermined, and the stoichiometry of the zinc: Aβ assemblies estimated.(FIG. 7 d) Following this procedure, the filters, retaining Zn: Aβassemblies, were washed with buffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH7.4)+EDTA (50 μM×300 μl, 700 g, 4 minutes). The amounts ofzinc-precipitated Aβ₁₋₄₀ resolubilized in the filtrate fraction weredetermined by OD₂₁₄, and expressed as a percentage of the amountoriginally retained by the respective filters. ⁶⁵Zn released into thefiltrate was measured by γ-counting.

Example 8 Zinc-induced Tinctorial Amyloid Formation

(FIG. 8 a) depicts Zinc-induced human Aβ₁₋₄₀ precipitate stained withCongo Red. The particle diameter is 40μ. Aβ₁₋₄₀ (200 μl×25 μM in buffer1 (100 mM NaCl, 20 mM Tris-HCl, pH 7.4)) was incubated (30 minutes, 37°C.) in the presence of 25 μM Zn²⁺. The mixture was then centrifuged(16,000 g×15 minutes), the pellet washed in buffer 1 (100 mM NaCl, 20 mMTris-HCl, pH 7.4)+EDTA (50 μM), pelleted again and resuspended in CongoRed (1% in 50% ethanol, 5 minutes). Unbound dye was removed, the pelletwashed with buffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH 7.4) and mountedfor microscopy. (FIG. 8 b) The same aggregate visualized under polarizedlight, manifesting green birefringence. The experiment was repeated withEDTA (50 μM) substituted for Zn²⁺ and yielded no visible material.

Example 9 Effect of Zinc and Copper Upon Human, ¹²⁵I-human and RatAβ₁₋₄₀ Aggregation into >0.2μ Particles

Stock human and rat Aβ₁₋₄₀ peptide solutions (16 μM) in water werepre-filtered (Spin-X, Costar, 0.2μ cellulose acetate, 700 g), brought to100 mM NaCl, 20 mM Tris-HCl, pH 7.4 (buffer 1)±EDTA (50 μM) or metalchloride salts, incubated (30 minutes, 37° C.) and then filtered again(700 g, 4 minutes). The fraction of the Aβ₁₋₄₀ in the filtrate wascalculated by the ratio of the filtrate OD₂₁₄ (the response of theOD₂₁₄, titrated against human and rat Aβ₁₋₄₀ concentrations (up to 20 μMin the buffers used in these experiments), was determined to be linear)relative to the OD₂₁₄ of the unfiltered sample. All data points are intriplicate, unless indicated. (FIG. 9) A graph showing the proportionsof Aβ₁₋₄₀, incubated±Zn²⁺ (25 μM) or Cu²⁺ or EDTA (50 μM) and thenfiltered through 0.2μ, titrated against peptide concentration.

Example 10 Effect of Zinc Upon Aβ Produced in Cell Culture

A cell culture, preferably mammalian cell culture, expressing,preferably overexpressing, human APP is established according towell-known methods in the art, e.g. N. Suzuki et al., Science264:1336-1340 (1994); X-D Cai et al., Science 259:514-516 (1993); F. S.Esch et al., Science 248:1122-1124 (1990). Next, zinc is added to theculture medium to final concentration from about 200 nM to about 5 μM.Then the cell cultures, containing zinc, are incubated from about 15minutes to as long as they can survive in the culture. Preferably, thecells are incubated for 3 to 4 days. While fresh media may be added tothe cultures, no spent medium should be taken out since it containsamyloid or zinc-induced Aβ aggregates.

The media which can be used are isotonic or physiological media, atphysiological pH (about 7.4). Preferably Tyrode's buffer is used withcalcium, magnesium, and potassium, as well as glucose. Any medium usedmust be devoid of cysteine, glutamate, aspartate, and histidine sincethese amino acids chelate zinc. Basically, any isotonic buffer orphysiological medium which minimizes constituents which chelate zinc maybe used. For example, Krebs Mammalian Ringer Solutions, in Data forBiochemical Research, 3d Edition by Dawson et al., Oxford SciencePublications, pp.446 (N.Y. 1986), and page 447 for Balanced SaltSolutions, provide recipes for making various useful media. Theconstituents that should be left out are serum and the four amino acidsmentioned above.

The cell culture should be incubated at about 37 degrees centigrade withair or O₂/CO₂ (the maximum concentration of CO₂ is 5%).

Next, the cells and the medium are harvested together. A detergent suchas Triton (at concentrations of about 1-2% v:v) is added and the mixtureis incubated for about 3 minutes to overnight. Preferably, however, itis incubated for about 1 to 2 hours.

After incubation, the cell debris as well as amyloid and zinc-induced Aβaggregates are pelleted by centrifugation. The pellet is suspended inpepsin (about 2%) or in any other peptidase, and it is incubated fromabout 1 hour to overnight to allow digestion of the cell debris.

Again, it is pelleted, washed with PBS or any other appropriate saltsolution, stained with Congo Red, washed again, pelleted to remove anyunbound Congo Red, and resuspended in aqueous solution. At this point, asample can be visually inspected under a microscope. Further, it can bequantitated using a grid.

Example 11 Assay for Predicting the Effectiveness of Candidate Reagentsin Cell Culture

The assay is set up in duplicate as described in Example 10. However, acandidate reagent is added to one of the two cell cultures and EDTA isadded to the other cell culture. After the final step in Example 10, theamount of amyloid and zinc-induced Aβ aggregates are compared under themicroscope. The probability and level of effectiveness of the candidatereagent is assessed based on the degree decrease in formation of amyloidand zinc-induced Aβ aggregates in the cell culture.

Example 12 Rapid Assay for Detection of Aβ Amyloid Formation inBiological Fluid

Cerebrospinal fluid (CSF) is obtained from a healthy human subject(control) and a human patient suspected of amyloidosis. Both samples ofCSF are titrated by serial dilutions, e.g., neat, 1:2, 1:4, 1:6, . . . ;dilutions may be made up to 1:10,000.

To each of the samples, an equal amount of Aβ peptide in water is addedto the final concentration of above about 10 μM, preferably about 10 toabout 25 μM.

Next, a solution which contains a heavy metal cation capable of bindingto a peptide comprising at least amino acids 6 to 28 of Aβ, preferablyZn²⁺, plus NaCl and a buffer, e.g., Tris at pH 7.4, is added to thefinal heavy metal cation, e.g., Zn²⁺, to a final concentration of aboutabove 300 nM, preferably 25 μM.

Then, the samples are centrifuged to form pellets. Pellets are stainedwith an amyloid-staining dye, e.g., Congo Red, and observed under amicroscope, thereby comparing levels of Aβ amyloid in the control versusthe sample from the patient with amyloidosis. If quantification ofamyloid is desired, a grid can be used.

Example 13 Rapid Assay for Detection of Aβ Amyloid Formation inBiological Fluid Using ³H-Aβ

The assay is set up as explained in Example 12, except that the Aβpeptide added is labelled beforehand by tritium. Moreover, aftercentrifugation, the pellets are counted in a scintillation counter.

The preferred method of detecting the amyloid, however, is by usingfiltration techniques as described above instead of centrifugation.After the samples are passed through a filter, the filters are added toscintillation fluid and the counts are determined.

Comparing the CPM from control samples with samples of the suspectedamyloidosis patient, it can be determined whether the patient is in factafflicted with amyloidosis. That is, an elevated CPM count in thepatient samples compared to the control samples is indicative ofamyloidosis.

Example 14 ELISA for Detection and/or Quantification of Aβ Peptides

Aβ-specific antibody of the enzyme-antibody conjugate binds to Aβpeptide bound to the heavy metal cation which is bound to the microtiterwell surface. The conjugated enzyme cleaves a substrate to generate acolored reaction product that can be detected spectrophotometrically.The absorbance of the colored solution in individual microtiter wells isproportional to the amount of Aβ peptide.

This assay is optimized for detection and quantitation of Aβ peptide inneat body fluids or in a partially purified or purified Aβ peptidepreparation.

Pretreatment of Samples Before ELISA

The body fluid or sample of partially purified Aβ may be treated priorto transfer to the 96-well plate to increase the efficiency of Aβabsorption to the solid-phase support. Treatments can include, but arenot restricted to: pre-incubation with methylating agents such asN-methyl malemide (1-10 mM for 1-2 hr) that disrupt protein metalbinding sites involving a cysteine residue (the Aβ peptide does notcontain a cysteine residue); the addition of soluble metal salts such assoluble MgCl₂ (0.5-5 mM) that block non-specific metal binding sites onproteins; the addition of compounds such as CuCl₂ (0.2-2 mM) which canchange the polymerization state of Aβ; and the addition of buffers toacidify the solution.

Materials

-   -   Aβ peptide (purified or partially purified) or neat body fluid        and controls (synthetic peptide standard)    -   Coating buffer (Tris 20 mM, pH 7.4, 150 mM NaCl)    -   Diluting buffer (Tris 20 mM, pH 7.4, 150 mM NaCl)    -   Blocking buffer (2% gelatine, Tris 20 mM, pH 8.0, 150 mM NaCl)    -   Wash buffer (Tris 20 mM, pH 8.0, 150 mM NaCl)    -   Normal saline (150 mM NaCl)    -   10 mM diethanolamine, pH 9.5, containing 0.5 mM MgCl₂    -   Urease-, HRPO-, or alkaline phosphatase-Aβ peptide conjugate        (prepared as described in UNIT 11.1 of Current Protocols in        Molecular Biology, Vol. 2, Ausubel et al., editors, (Greene        Publishing Associates and Wiley-Interscience, publishers), New        York.    -   Urease substrate solution (Allelix #1001 100), peroxidase        substrate solution (Kirkegaard and Perry #50-62-00), or alkaline        phosphatase substrate solution    -   Zinc (Xenopore ZCP 00100) or Copper (Xenopore CCP 00100)    -   96-well microtiter plates (or other heavy metal cation bound        plates as described in Materials and Methods)    -   Multichannel pipet    -   Adhesive covers or tape for covering microtiter plates    -   Microtiter plate spectrophotometer with 590-nm and/or 405-run        filters

-   1. Dissolve purified or partially purified Aβ peptide and controls    in coating buffer at about 0.2-2.0 μg/ml.    -   Depending on the affinity of the antibody for the Aβ peptide, it        may be necessary to increase or decrease the amount of Aβ        peptide or neat body fluid in coating buffer.    -   For specificity testing, include closely related control        antigens which the antibody should not recognize.

-   2. Fill columns 2 through 12 of a 96-well microtiter plate with 0.1    ml coating buffer.    -   A 96 well plate is divided into 12 columns (labeled 1-12) and 8        rows (labeled A-H).

-   3. Starting in column 1 of a 96-well microtiter plate, serially    dilute Aβ peptide in coating buffer. Place 0.2 ml of Aβ peptide    solution in each well in column 1. Remove 0.1 ml from each well with    a multichannel pipet and transfer to each well in column 2, which    contains 0.1 ml of coating buffer. Pipet material in column 2 five    times up and down. Remove 0.1 ml from each well in column 2 and    transfer to column 3. Repeat this procedure through column 11.    Remove 0.1 ml from column 11 and discard. Leave column 12 blank.    Prepare two identical plates for duplicate assays. For controls    prepare plates as per steps 2 and 3, using control material in place    of Aβ solution. An example of a control material is a coating buffer    without Aβ peptide.    -   This will give a range of dilutions in each of 8 rows (A to H)        from 1:1 through 1:1,024 [i.e., column number (dilution); 1        (1:1), 2 (1:2), 3 (1:4), 4 (1:8), 5 (1:16), 6 (1:32), 7 (1:64),        8 (1:128), 9 (1:256), 10 (1:512), and 11 (1:1,024)].

-   4. Cover the plates with adhesive covers or tape and incubate for 2    hours at 37° C.

-   5. Remove Aβ peptide solution by shaking into a sink and fill all    wells with 0.3 ml blocking buffer. Incubate for 2 h at 37° C.    -   HRPO is inactivated by sodium azide. Do not use buffers        containing sodium azide with HRPO-antibody conjugates.    -   Filter sterilize buffers used routinely (i.e., diluting buffer)        and store at 4° C.

-   6. Remove blocking buffer by shaking into a sink and add 0.3 ml of    washing buffer. Empty wells and refill with washing buffer. Repeat    one more time.

-   7. Remove washing buffer by shaking into a sink and then fill rows B    to H with 0.1 ml diluting buffer.

-   8. Add 0.2 ml of enzyme-antibody conjugate diluted in diluting    buffer to row A of each plate. Recommended starting dilution of    conjugate is 1:100. Serially dilute conjugate from row A to row H by    transferring 0.1 ml to the well in the next row, as described in    step 3. Final volume of conjugate in each well should be 0.1 ml.    -   This will give a range of dilution from 1:100 through 1:12,800        [row (dilution): A (1:100, B (1:200), C (1:400), D (1:800), E        (1:1,600), F (1:3,200), G (1.:6,400), and H (1:12,800)].

-   9. Cover the plates with adhesive covers or tape and incubate for a    set length of time at a controlled temperature.    -   Time and temperature of incubation are determined empirically.        Generally, 30 to 90 min at 37° C. is sufficient. Longer times of        incubation may increase sensitivity, but nonspecific binding may        also increase. An example is the monoclonal mouse antibody 6E10.        For example, the mouse mAb 6E10 has an optimal incubation of 2 h        at 37° C. or overnight at room temperature (18-20° C.).

-   10. Shake out the plates into a sink. Wash plates with wash buffer    twice (0.3 ml each time) for urease- or alkaline    phosphatase-antibody conjugates and four times for HRPO-antibody    conjugates by filling well and shaking out the wash buffer into a    sink. If an urease-antibody conjugate was used, rinse plates an    additional three times with normal saline. If an alkaline    phosphatase-antibody conjugate was used, rinse plates twice with 10    mM diethanolamine, pH 9.5, containing 0.5 mM MgCl₂. Pat plates dry    by inverting on a paper towel.

-   11. Add 0.2 ml of either urease, peroxidase, or alkaline phosphatase    substrate solution, depending on the enzyme-antibody conjugate used.    For example, a high sensitivity substrate for HRPO is a TMB solution    (Pierce catalog no. 34024) and that is measured at 450 nm.    Appropriate absorbances include 590 nm (urease), 405 nm (HRPO or    alkaline phosphatase), and 450 (HRPO when TMB is used as the    substrate) using a microtiter plate spectrophotometer.    -   For alkaline phosphatase-based assays, add 100 μl of 0.1 M EDTA        to each well at the end of the incubation in order to stop the        reaction. For HPRO assays using TMB substrate solutions, the        reaction is terminated after incubation by the addition of 25 μl        of sulfuric acid (1-3 M).

-   12. Plot absorbance versus (Aβ) antigen concentration [Ag] on    semilog paper for analysis of each dilution of enzyme-antibody    conjugate. For working dilution of conjugate, choose a concentration    that provides maximum sensitivity over a linear range of [Ag] and    minimum binding (below 0.05 absorbance units) to control antigens    (synthetic peptide standards).

-   13. Serially dilute individual body fluid and controls or partially    purified or purified Aβ peptide preparations, as described in    step 3. Use two columns per sample.

-   14. Repeat steps 4-6.

-   15. Shake out diluting buffer into a sink and add 0.1 ml per well of    enzyme-antibody conjugate diluted in diluting buffer at the    concentration determined in step 12.

-   16. Cover the plates with adhesive covers and incubate under the    same conditions as used in step 9.

-   17. Repeat steps 10 and 11.

-   18. Compare the absorbance of the unknown to the standard curve for    the enzyme-antibody conjugate concentration that was plotted in step    12 in order to determine the quantity of antigen expressed per    volume of a body fluid or a sample of partially purified Aβ peptide.

Example 15 A Method for Bulk Purification of Aβ Peptide from BiologicalFluids

The bulk purification of Aβ from biological fluids is best achieved withcopper charged chelating-Sepharose (Pharmacia catalog no. 17-057501).The cysteine groups in the sample proteins are first methylated with amaleimide compound (e.g., N-methyl maleimide (Sigma catalog no.930-88-1), about 1-10 mM, about 1 hour; also see Yomomote and KeKine,Anal. Biochem. 90:300-308 (1978)).

The methylated sample is then acidified by titrating pH to about 5.0using about 1M sodium acetate, pH about 3.5, and the total NaClconcentration increased by about 500 mM with about 5M NaCl. The pH ofthe sample is monitored with a glass pH detector or pH indicator paperwhile sodium acetate is added dropwise with gentle stirring until therequired pH is obtained.

The sample is then applied to a copper-charged chelating-Sepharosecolumn (e.g., about 250 μl bed volume for about 15 ml of CSF) asdescribed above, in the section entitled Experimental Procedures.Equilibration buffer is about 500 mM NaCl about 50 mM MES pH about 5.0and is used to wash the column. The Sepharose can be developed withabout 500 mM NaCl, 50 mM EDTA, pH 8.0 alone and the eluate sampled forwestern blot analysis. The treatment of 15 ml of CSF by this methodenriched both soluble APP as well as 4.3 and 3.6 kD a species of Aβ(identified by an antibody that identifies an epitope in the first 16residues of Aβ; commercially available).

In order to bind copper or zinc, the peptide requires an intact domainfrom residues 6-28. 4G8 only recognized the two Aβ species and not APP,confirming that the APP captured by the Sepharose was post-secretasecleaved soluble APP. The use of specific anti-Aβ antibodies as describedabove on western blot analysis of these products can confirm thespecificity of the ELISA immunoreactivity.

Example 16 A Method for Purification of Aβ Peptide when the Volume ofthe Biological Material is Less than about 4 ml

The cysteine groups in the sample proteins are first methylated with amaleimide compound (e.g., N-methyl maleimide (Sigma catalog no.930-88-1) about 1-10 mM, about 1 hour; also see Yomomote and KeKine,Anal. Biochem. 90:300-308 (1978)).

The methylated sample is then acidified by titrating pH to about 5.0using about 1M sodium acetate, pH about 3.5, and the total NaClconcentration increased by about 500 mM with about 5M NaCl. The pH ofthe sample is monitored with a glass pH detector or pH indicator paperwhile sodium acetate is added dropwise with gentle stirring until therequired pH is obtained.

Free copper-charged chelating-Sepharose slurry (about 60 μl of about 50%v/v) is added to the sample.

Following centrifugation (preferably, low speed centrifugation (about1,500 g, for about 3 minutes, equilibration buffer is used to wash theSepharose pellet. Equilibration buffer is about 500 mM NaCl about 50 mMMES, pH about 5.0.

The Sepharose can be developed (protein is eluted by the addition of theeluting buffer) with about 500 mM NaCl, 50 mM EDTA, pH 8.0 alone and theeluate sampled for western blot analysis.

Having now fully described this invention, it will be understood bythose of skill in the art that it can be performed within any wide rangeof equivalent modes of operation as well as other parameters withoutaffecting the scope of the invention or any embodiment thereof.

All patents and publications cited in the present specification areincorporated by reference herein in their entirety.

1. An assay for detecting or quantifying Aβ peptide which may be presentin a candidate solution, comprising: (a) contacting the candidatesolution with a solid support with a heavy metal cation immobilizedthereon to capture Aβ peptide on the surface of the solid support,thereby forming a first complex which comprises solid support/heavymetal cation/Aβ peptide; (b) blocking all exposed metal binding sitesremaining after Aβ capture with a blocker; (c) contacting the firstcomplex, which has been passed through step (b), with a polyclonalantibody specific for Aβ peptide to form a second complex whichcomprises solid support/heavy metal cation/Aβ peptide/polyclonalantibody specific for Aβ peptide; (d) labelling the second complex toform a detectable third complex which comprises solid support/heavymetal cation/Aβ peptide peptide/polyclonal antibody specific for Aβpeptide/label; and (e) detecting the third complex, and quantifying Aβpeptide which may be present in the candidate solution.
 2. An assay fordetecting or quantifying Aβ peptide which may be present in a candidatesolution, comprising: (a) contacting the candidate solution with a solidsupport with a heavy metal cation immobilized thereon to capture Aβpeptide on the surface of the solid support, thereby forming a firstcomplex which comprises solid support/heavy metal cation/Aβ peptide; (b)blocking all exposed metal binding sites remaining after Aβ capture witha blocker; (c) contacting the first complex, which has been passedthrough step (b), with a polyclonal antibody specific for Aβ peptide,called Aβ antibody, to form a second complex which comprises solidsupport/heavy metal cation/Aβ peptide/Aβ antibody; (d) contacting saidsecond complex with one or more anti-antibodies specific to the Aβantibody to form a third complex which comprises solid support/heavymetal cation/Aβ peptide/Aβ antibody/one or more anti-antibodies; (e)labelling said third complex to form a detectable fourth complex whichcomprises solid support/heavy metal cation/Aβ peptide/Aβ antibody/one ormore anti-antibodies/label; and (f) detecting the fourth complex,thereby quantifying Aβ peptide which may be present in the candidatesolution.
 3. The assay as claimed in claim 1, wherein said heavy metalcation is selected from the group consisting of zinc (II) and copper(II) complexed to nitriloacetic acid.
 4. The assay as claimed in claim2, wherein said heavy metal cation is selected from the group consistingof zinc (II) and copper (II) complexed to nitriloacetic acid.
 5. A kitfor canying out the assay of claim 1 or 2, which comprises a carriermeans compartmentalized in close confinement therein to receive one ormore container means which comprises a first container means containinga solid support having a heavy metal cation immobilized thereon and asecond container means containing a polyclonal antibody specific for Aβpeptide.
 6. The kit as claimed in claim 5, wherein said heavy metalcation is selected from the group consisting of zinc (II) and copper(II) complexed to nitriloacetic acid.
 7. The kit as claimed in claim 5,wherein said antibody is labeled with a radioisotope.
 8. The kit asclaimed in claim 5, wherein said antibody is labeled with an enzyme. 9.The kit as claimed in claim 5, wherein said carrier means furthercomprises a third container means containing an anti-antibody which isspecific for the Aβ antibody.
 10. The kit as claimed in claim 9, whereinsaid anti-antibody is labeled with a radioisotope.
 11. A kit forcarrying out the assay of claim 1 or 2, which comprises a carrier meanscompartmentalized in close confinement therein to receive one or morecontainer means which comprises a first container means containing asolid support having a heavy metal cation immobilized thereon and asecond container means containing a labelled polyclonal antibodyspecific for Aβ peptide.
 12. The kit as claimed in claim 11, whereinsaid heavy metal cation is selected from the group consisting of zinc(II) and copper (II) complexed to nitriloacetic acid.
 13. The kit asclaimed in claim 11, wherein the labeled antibody is labeled by aradioisotope.
 14. The kit as claimed in claim 11, wherein said antibodyis labeled with an enzyme.
 15. A kit for carrying out the assay of claim1 or 2, which comprises a carrier means compartmentalized in closeconfinement therein to receive one or more container means whichcomprises a first container means containing a solid support having aheavy metal cation immobilized thereon and a second container meanscontaining a polyclonal antibody specific for Aβ peptide bound to alabelled anti-antibody.
 16. The kit as claimed in claim 15, wherein saidheavy metal cation is selected from the group consisting of zinc (II)and copper (II) complexed to nitriloacetic acid.
 17. The kit as claimedin claim 15, wherein the labeled antibody is labeled by a radioisotope.18. The kit as claimed in claim 15, wherein said labeled antibody islabeled with an enzyme.
 19. The kit as claimed in claim 8, wherein saidenzyme is horseradish peroxidase.
 20. The kit as claimed in claim 14,wherein said enzyme is horseradish peroxidase.
 21. The kit as claimed inclaim 18, wherein said enzyme is horseradish peroxidase.